Polynucleotides encoding chemokine β-6 antagonists

ABSTRACT

Human chemokine β-6 agonist and antagonist polypeptides and DNA encoding such polypeptides and a procedure for producing such polypeptides by recombinant techniques are disclosed. The chemokine β-6 antagonists of the present invention may be employed to treat rheumatoid arthritis, lung inflammation, allergy, asmtha, infectious diseases and to prevent inflammation and atherosclerosis. The chemokine β-6 agonists may be employed to myeloprotect patients undergoing chemotherapy.

This application is a divisional of application Ser. No. 08/995,156,filed Dec. 19, 1997, U.S. Pat. No. 6,028,169, which claims the benefitof Provisional Application No. 60/042,269, filed Mar. 31, 1997, both ofwhich disclosures are herein incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to human chemokine β-6 agonist andantagonist polypeptides and DNA (RNA) encoding such polypeptides and aprocedure for producing such polypeptides by recombinant techniques. Thechemokine β-6 antagonists of the present invention may be employed totreat rheumatoid arthritis, lung inflammation, allergy, asthma,infectious diseases and to prevent inflammation and atherosclerosis. Thechemokine β-6 agonists may be employed to myeloprotect patientsundergoing chemotherapy. Chemokine β-6 (Ckβ-6) is also referred toherein as MPIF-2 and eotaxin-2.

2. Related Art

There are three forms of monocyte chemotactic protein, namely, MCP-1,MCP-2 and MCP-3. All of these proteins have been structurally andfunctionally characterized and have also been cloned and expressed.MCP-1 and MCP-2 have the ability to attract leukocytes (monocytes, andleukocytes), while MCP-3 also attracts eosinophils and T lymphocytes(Dahinderi, E., et al., J. Exp. Med. 179:751-756 (1994)).

Initially, human monocyte-specific attracting factor, was purified froma glioma cell line and a monocytic cell line. Matsushima, K., et al, J.Exp. Med. 169:1485-1490 (1989). This factor was originally designatedglioma-derived chemotactic factor (GDCF) and monocyte chemotactic andactivating factor (MCAF) by Matsushima, et al. This factor is nowreferred to as MCP-1. Subsequent cloning of the cDNA for MCP-1 showed itto be highly similar to the murine JE gene. The JE gene could bemassively induced in murine fibroblasts by platelet-derived growthfactor. Cochran, B.H., et al, Cell 33:939-947 (1983). Murine JE ishighly similar to MCP-1. The MCP-1 protein is 62% identical to murine JEin a region of 68 shared N-terminal residues. It is widely accepted thatJE and MCP-1 are species homologs.

A method of suppressing tumor formation in a vertebrate by administeringJE/MCP-1 has been disclosed in PCT application WO-92/20372, along withmethods of treating localized complications of malignancies and methodsof combatting parasitic infection by administering JE/MCP-1. Expressionof the JE/MCP-1 protein in malignant cells was found to suppress thecells ability to form tumors in vivo.

Human MCP-1 is a basic peptide of 76 amino acids with a predictedmolecular mass of 8,700 daltons. MCP-1 is inducibly expressed mainly inmonocytes, endothelial cells and fibroblasts. Leonard, E. J. andYoshimura, T., Immunol. Today 11:97-101 (1990). The factors which inducethis expression is IL-1, TNF or lipopolysaccharide treatment.

Other properties of MCP-1 include the ability to strongly activatemature human basophils in a pertussis toxin-sensitive manner. MCP-1 is acytokine capable of directly inducing histamine release by basophils,(Bischoff, S. C., et al., J. Exp. Med. 175:1271-1275 (1992)).Furthermore, MCP-1 promotes the formation of leukotriene C4 by basophilspretreated with Interleukin 3, Interleukin 5, or granulocyte/macrophagecolony-stimulating factor. MCP-1 induced basophil mediator release mayplay an important role in allergic inflammation and other pathologiesexpressing MCP-1.

Clones having a nucleotide sequence encoding a human monocytechemotactic and activating factor (MCAF) reveal the primary structure ofthe MCAF polypeptide to be composed of a putative signal peptidesequence of 23 amino acid residues and a mature MCAF sequence of 76amino acid residues. Furutani, Y. H., et al., Biochem. Biophys. Res.Commu. 159:249-55 (1989). The complete amino acid sequence of humanglioma-derived monocyte chemotactic factor (GDCF-2) has also beendetermined. This peptide attracts human monocytes but not neutrophils.It was established that GDCF-2 comprises 76 amino acid residues. Thepeptide chain contains 4 half-eysteines, at positions 11, 12, 36 and 52,which create a pair of loops, clustered at the disulfide bridges.Further, the MCP-1 gene has been designated to human chromosome 17.Mehrabian, M. R., et al., Genomics 9:200-3 (1991).

Certain data suggests that a potential role for MCP-1 is mediatingmonocytic infiltration of the artery wall. Monocytes appear to becentral to atherogenesis both as the progenitors of foam cells and as apotential source of growth factors mediating intimal hyperplasia.Nelken, N. A., et al., J. Clin. Invest. 88:1121-7 (1991). It has alsobeen found that synovial production of MCP-1 may play an important rolein the recruitment of mononuclear phagocytes during inflammationassociated with rheumatoid arthritis and that synovial tissuemacrophages are the dominant source of this cytokine. MCP-1 levels werefound to be significantly higher in synovial fluid from rheumatoidarthritis patients compared to synovial fluid from osteoarthritispatients or from patients with other arthritides. Koch, A. E., et al.,J. Clin. Invest. 90:772-9 (1992).

MCP-2 and MCP-3 are classified in a subfamily of proinflammatoryproteins and are functionally related to MCP-1 because they specificallyattract monocytes, but not neutrophils. Van Damme, J., et al., J. Exp.Med. 176:59-65 (1992). MCP-3 shows 71% and 58% amino acid homology toMCP-1 and MCP-2 respectively. MCP-3 is an inflammatory cytokine thatregulates macrophage functions.

The transplantation of hemolymphopoietic stem cells has been proposed inthe treatment of cancer and hematological disorders. Many studiesdemonstrate that transplantation of hematopoietic stem cells harvestedfrom the peripheral blood has advantages over the transplantation ofmarrow-derived stem cells. Due to the low number of circulating stemcells, there is a need for induction of pluripotent marrow stem cellmobilization into the peripheral blood. Reducing the amount of blood tobe processed to obtain an adequate amount of stem cells would increasethe use of autotransplantation procedures and eliminate the risk ofgraph versus host reaction connected with allotransplantation.Presently, blood mobilization of marrow CD34⁺ stem cells is obtained bythe injection of a combination of agents, including antiblastic drugsand G-CSF or GM-CSF. Drugs which are capable of stem cell mobilizationinclude IL-1, IL-7, IL-8, and NIP-1a Both IL-1 and IL-8 demonstrateproinflammatory activity that may be dangerous for good engrafting. IL-7must be administered at high doses over a long duration and MIP-1 a isnot very active as a single agent and shows best activity when incombination with G-CSF.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, there isprovided a novel full-length or mature polypeptide, as well asbiologically active, diagnostically or therapeutically useful fragments,analogs and derivatives thereof. The polypeptide of the presentinvention is of human origin.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules encoding a polypeptide of thepresent invention including mRNAs, DNAs, cDNAs, genomic DNAs as well asanalogs and biologically active and diagnostically or therapeuticallyuseful fragments, analogs and derivatives thereof.

The present invention also provides isolated nucleic acid moleculescomprising a polynucleotide encoding the Ckβ-6 polypeptide having theamino acid sequence shown in FIG. 1 (SEQ ID NO:2) or the amino acidsequence encoded by the cDNA clone deposited as ATCC Deposit No. 75703on Mar. 10, 1994. The nucleotide sequence determined, at least in part,by sequencing the deposited Ckβ-6 clone, which is shown in FIG. 1 (SEQID NO:1), contains an open reading frame encoding a polypeptide of 119amino acid residues, with a leader of about 26 amino acid residues. Theamino acid sequence of the mature Ckβ-6 protein is shown in FIG. 1, asamino acid residues 1-93 of SEQ ID NO:2.

Thus, one aspect of the invention provides an isolated nucleic acidmolecule comprising a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding an Ckβ-6 polypeptide having the complete amino acid sequence inFIG. 1 (SEQ ID NO:2); (b) a nucleotide sequence encoding an Ckβ-6polypeptide having the complete amino acid sequence in FIG. 1 (SEQ IDNO:2) excepting the N-terminal methionine; (c) a nucleotide sequenceencoding an Ckβ-6 polypeptide having the amino acid sequence atpositions 1-93 in FIG. 1 (SEQ ID NO:2); (d) a nucleotide sequenceencoding the Ckβ-6 polypeptide having the complete amino acid sequenceencoded by the cDNA clone contained in ATCC Deposit No. 75703; (e) anucleotide sequence encoding the Ckβ-6 polypeptide having the completeamino acid sequence encoded by the cDNA clone contained in ATCC DepositNo. 75703 excepting the N-terminal methionine; (f) a nucleotide sequenceencoding the mature Ckβ-6 polypeptide having the amino acid sequenceencoded by the cDNA clone contained in ATCC Deposit No. 75703; and (g) anucleotide sequence complementary to any of the nucleotide sequences in(a), (b), (c), (d), (e) or (f) above.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode fragments, analogs andderivatives of the Ckβ6 polypeptide having the deduced amino acidsequence of FIG. 1 (SEQ ID NO:2) or the polypeptides encoded by the cDNAof the deposited clone. The variants of the polynucleotides can be anaturally occurring allelic variant of the polynucleotides or anon-naturally ocurring variant of the polynucleotides.

Further embodiments of the invention include islolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 90% homologous or identical, and more preferably at least 95%,96%, 97%, 98%, or 99% identical, to any of the nucleotide sequences in(a), (b), (c), (d), (e), (f) or (g), above, or a polynucleotide whichhybridizes under stringent hybridization conditions to a polynucleotidein (a), (b), (c), (d), (e), (f) or (g), above. These polynucleotideswhich hybridize do not hybridize under stringent hybridizationconditions to a polynucleotide having a nucleotide sequence consistingof only A residues or of only T residues.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells.

In accordance with yet a further aspect of the present invention, thereis provided a process for producing such polypeptide by recombinanttechniques comprising culturing recombinant prokaryotic and/oreukaryotic host cells, containing a nucleic acid sequence encoding apolypeptide of the present invention, under conditions promotingexpression of said protein and subsequent recovery of said protein.

The invention further provides an isolated Ckβ-6 polypeptide having anamino acid sequence selected from the group consisting of: (a) the aminoacid sequence of the Ckβ-6 polypeptide having the complete amino acidsequence, including the leader sequence shown in FIG. 1 (SEQ ID NO:2);(b) the amino acid sequence of the Ckβ-6 polypeptide having the completeamino acid sequence, including the leader sequence shown in FIG. 1,excepting the N-terminal methionine; (c) the amino acid sequence of themature Ckβ-6 polypeptide (without the leader) having the amino acidsequence at positions 1-93 in FIG. 1 (SEQ ID NO:2); (d) the amino acidsequence of the Ckβ-6 polypeptide having the complete amino acidsequence, including the leader sequence, encoded by the cDNA clonecontained in ATCC Deposit No. 75703; (e) the amino acid sequence of theCkβ6 polypeptide having the complete amino acid sequence, including theleader sequence, encoded by the cDNA clone contained in ATCC Deposit No.75703, excepting the N-terminal methionine; and (f) the amino acidsequence of the mature Ckβ-6 polypeptide having the amino acid sequenceencoded by the cDNA clone contained in ATCC Deposit No. 75703.

Polypeptides of the present invention also include homologouspolypeptides having an amino acid sequence with at least 90% identity,and more preferably at least 95% identity to those described in (a),(b), (c), (d), (e) or (f) above, as well as polypeptides having an aminoacid sequence at least 80% identical, more preferably at least 90%identical, and still more preferably 95%, 96%, 97%, 98% or 99% identicalto those above.

An additional embodiment of this aspect of the invention relates to apeptide or polypeptide which has the amino acid sequence of an epitopebearing portion of an Ckβ-6 polypeptide having an amino acid sequencedescribed in (a), (b), (c), (d), (e) or (f) above. Peptides orpolypeptides having the amino acid sequence of an epitope bearingportion of an Ckβ-6 polypeptide of the invention include portions of anCkβ-6 polypeptide with at least six or seven, preferably at least nine,and more preferably at least about 30 amino acids to about 50 aminoacids, although epitope-bearing polypeptides of any length up to andincluding the entire amino acid sequence of a polypeptide of theinvention described above also are included in the invention.

An additional nucleic acid embodiment of the invention relates to anisolated nucleic acid molecule comprising a polynucleotide which encodesthe amino acid sequence of an epitope-bearing portion of an Ckβ-6polypeptide having an amino acid sequence in (a), (b), (c), (d), (e) or(f), above.

The present invention also provides, in another aspect, pharmaceuticalcompositions comprising an Ckβ-6 polynucleotide, probe, vector, hostcell, polypeptide, fragment, variant, derivative, epitope bearingportion, antibody, antagonist or agonist.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptide, or polynucleotideencoding such polypeptide for therapeutic purposes, for example, forstem cell mobilization, myeloprotection and neuronal protection, totreat tumors, to promote wound healing, to combat parasitic infectionand to regulate hematopoiesis.

An additional aspect of the invention is related to a method fortreating an individual in need of an increased level of Ckβ-6 activityin the body comprising administering to such an individual a compositioncomprising a therapeutically effective amount of an isolated Ckβ-6polypeptide of the invention or an agonist thereof.

A still further aspect of the invention is related to a method fortreating an individual in need of a decreased level of Ckβ-6 activity inthe body comprising, administering to such an individual a compositioncomprising a therapeutically effective amount of an Ckβ-6 antagonist.Preferred antagonists for use in the present invention are Ckβ-6specific or CCR3 receptor specific antibodies.

In accordance with yet a further aspect of the present invention, thereare provided antibodies against such polypeptides. In anotherembodiment, the invention provides an isolated antibody that bindsspecifically to an Ckβ-6 polypeptide having an amino acid sequencedescribed in (a), (b), (c), (d), (e) or (f) above.

The invention further provides methods for isolating antibodies thatbind specifically to an Ckβ-6 polypeptide having an amino acid sequenceas described herein. Such antibodies are useful diagnostically ortherapeutically as described below.

In accordance with another aspect of the present invention, there areprovided agonists which mimic the polypeptide of the present inventionand bind to receptors to elicit second messenger responses.

In accordance with yet another aspect of the present invention, thereare provided antagonists to such polypeptides, which may be used toinhibit the action of such polypeptides, for example, in the treatmentof rheumatoid arthritis, lung inflammation, histamine-mediated allergicreactions, infectious diseases, hyper-eosinophilic syndromes, silicosis,sarcoidosis and to prevent auto immune and chronic inflammation andatherosclerosis. Alternatively, such polypetides can be used to inhibitproduction of IL-1 and TNFα, to treat aplastic anemia, myelodysplasticsyndrome, asthma and arthritis.

In accordance with yet a further aspect of the present invention, thereis also provided nucleic acid probes comprising nucleic acid moleculesof sufficient length to specifically hybridize to a nucleic acidsequence of the present invention.

In accordance with still another aspect of the present invention, thereare provided diagnostic assays for detecting diseases relating tounderexpression or overexpression of the polypeptides and for detectingsusceptibility to diseases related to mutations in the nucleic acidsequences encoding a polypeptide of the present invention.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, for example, synthesis of DNA andmanufacture of DNA vectors, for the purpose of developing therapeuticsand diagnostics for the treatment of human disease.

The present invention also provides a screening method for identifyingcompounds capable of enhancing or inhibiting a cellular response inducedby an Ckβ-6 polypetide, which involves contacting cells which expressthe Ckβ-6 polypeptide with the candidate compound, assaying a cellularresponse, and comparing the cellular response to a standard cellularresponse, the standard being assayed when contact is made in absence ofthe candidate compound; whereby, an increased cellular response over thestandard indicates that the compound is an agonist and a decreasedcellular response over the standard indicates that the compound is anantagonist.

For a number of disorders, it is believed that significantly higher orlower levels of Ckβ-6 gene expression can be detected in certain tissuesor bodily fluids (e.g., serum, plama, urine, synovial fluid or spinalfluid) taken from an individual having such a disorder, relative to astandard Ckβ-6 gene expression level; i.e., the Ckβ-6 expression levelin tissue or bodily fluids from an individual not having the disorder,which involves: (a) assaying the Ckβ-6 gene expression level in cells orbody fluid of an individual; (b) comparing the Ckβ-6 gene expressionlevel with a standard Ckβ-6 gene expression level, whereby an increaseor decrease in the assayed Ckβ-6 gene expression level compared to thestandard expression level is indicative of a disorder. Such disordersinclude leukemia, chronic inflammation, autoimmune diseases, and solidtumors.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIG. 1 depicts the cDNA sequence (SEQ ID NO:1) and correspondingdetermined amino acid sequence (SEQ ID NO:2) of Ckβ-6. The 119 aminoacid sequence shown is the full length protein, with approximately thefirst 26 amino acids representing a leader sequence (underlined) suchthat the mature form of the protein is 93 amino acids in length. Thestandard one letter abbreviation for amino acids is used.

FIG. 2 illustrates a comparison of the amino acid sequence homologybetween the polypeptide of the present invention with human MCP-1 (SEQID NO:5). Ckβ-6 shows 36% identity and 52% similarity with human MCP-1as determined by the computer program Bestfit.

FIG. 3 illustrates the chemotactic activity of the polypeptide of thepresent invention on neutrophils (PMN) and peripheral blood mononuclearcells (PBMC). Neutrophils and peripheral blood mononuclear cells wereisolated from peripheral blood, loaded with calcein-AM and used forchemocaxis in a 96 well, single-use Neuroprobe chemotactic chamber.After 90 minutes incubation with Ckβ-6, the chamber was dismounted, thefilter air-dried and the number of cells which migrated through themembrane quantitated in a cytofluor II.

FIGS. 4A and 4B illustrates that Ckβ-6 inhibits the growth anddifferentiation of high proliferative potential colony forming cells(HPP-CFC) (A) and is not effective on low proliferative potential colonyforming cells (LPP-CFC) (B). In these experiments, 1,500 cells from lowdensity, non-adherent bone marrow cells were plated in agar-mediumsupplemented with 5 ng/ml mouse IL-3, 100 ng/ml mouse SCF, 10 ng/mlmouse IL-1a, 5 ng/ml human M-CSF, and with or without the indicatedconcentrations of Ckβ-6. Colonies were scored after 14 days ofincubation. Three experiments were performed. The results are presentedas mean number of colonies±SD. An irrelevant protein had no effects.

FIGS. 5A and 5B shows the effect of Ckβ-6 on bone marrow cells whichwere enriched in the primitive Lin− cells by removing committedprecursor cells (antibodies anti-CD11b, CD4, CD8, CD45R and Gr.-1). Thepanel A shows ratios±SD of LPP-CFC/HPP-CFC in the bone marrow cells(column 1) or Lin− cells (column 2) plated in agar-medium with 5 ng/mlIL-3, 100 ng/ml SCF, 10 ng/ml IL-1a, 5 ng/ml M-CSF. Columns 3, 4 and 5show the ratio of LPP-CFC/HPP-CFC found in the Lin− cells that werecultured with 5 ng/ml IL-3 and 100 ng/ml SCF (column 3), IL-3, SCF and50 ng/ml Ckβ-6 (column 4) or IL-3, SCF and 50 ng/ml of an irrelevantprotein (column 5). After 6 days, cultures were assayed for HPP-CFC andLPP-CFC. The panel B shows the cellularity after 6 days incubation.

FIG. 6 illustrates that Ckβ-6 protects HPP-CFC but not LPP-CFC from thecytotoxic effect of cytosine arabinoside (Ara-C) in vitro.

FIG. 7 illustrates that Ckβ-6 protects HPP-CFC but not LPP-CFC from thecytotoxic effect of 5-Fluorouracil (5-FU) in vitro.

FIG. 8 illustrates the effect of Ckβ-6 and Basic FGF on CorticalNeuronal Survival.

FIG. 9 illustrates the effect of Ckβ-6 and other chemokines on therelease of calcium by human eosinophils.

FIGS. 10A and 10B illustrates the ability of Ckβ-6 to act as achemoattractant for human eosinophils and basophils in-vitro. Also,illustrated is the ability of a monoclonal antibody directed against theCCR3 receptor (anti-CCR3) to block the migratory response of these celltypes.

FIGS. 11A-11D illustrate the effect of Ckβ-6 on histamine and LTC4release from human eosinophils and the ability of anti-CCR3 to blocksuch activity.

FIG. 12 illustrates the ability of Ckβ-6 to act as a chemoataactantin-vivo.

FIG. 13 shows an analysis of the Ckβ-6 amino acid sequence. Alpha, beta,turn and coil regions; hydrophilicity and hydrophobicity; amphipathicregions; flexible regions; antigenic index and surface probability areshown. In the “Antigenic Index-Jameson-Wolf” graph, the positive peaksindicate locations of the highly antigenic regions of the Ckβ-6 protein,i.e., regions from which epitope-bearing peptides of the invention canbe obtained.

FIGS. 14A and 14B demonstrates that HG00604 and HG00605 are agonist foreosinophils but HG00606 and HG00608 are not. Eosinophils were used forcalcium flux assays as described in Example 9. The various chemokines,including Eotaxin, were used at the concentrations indicated. Panel Aand B show the results obtained with two individual donors.

FIGS. 15A and B illustrates that HG00606 is an antagonist of HG00604 butHG00608 is not. Eosinophils were used for calcium flux assays asdescribed in Example 9. HG00604 was used in 10, 100, and 1000 ng/ml withor without 1000 ng/ml of HG00606 or HG00608. As shown, the presence ofHG00606 reduced the calcium flux directed by 10 or 100 ng/ml of HG00604.Under the same conditions, HG00608 showed no effects. Panel A and B showthe results obtained with two individual donors.

FIGS. 16A and 16B demonstrates that HG00606 is an antagonist of HG00604,Eotaxin and CkBeta-10. Eosinophils were used for calcium flux assays asdescribed in Example 9. HG00604, eotaxin and CkBeta-10 was used at 10,100, and 1000 ng/ml with or without 1000 ng/ml of HG00606. As shown thepresence of HG00606 reduced the calcium flux directed by 10 or 100 ng/mlof HG00604 or CkBeta-10 and the calcium flux directed by 10 ng/ml ofEotaxin. Panel A and B show the results obtained with two individualdonors.

FIG. 17 illustrates that HG00603 but not HG00606 is chemotactic foreosinophils. Eosinophils were used for chemotaxis assays as described inExample 13. Chemotaxis in response to Eotaxin (closed circles), HG00603(closed squares), or HG00606 (open triangles) is depicted as thechemotactic index and represents the average of 5 to 7 separateexperiments where individual experiments were performed in triplicate.

FIGS. 18A and 18B illustrates that HG00606 acts as an antagonist ofHG00603. Eosinophils were used for chemotaxis assays as described inExample 13. Chemotaxis in response to HG00603 (closed circles) orHG00603+HG00606 (closed diamonds) is depicted as the chemotactic indexfrom one representative experiment performed in triplicate. HG00603 wasadded to the bottom well of the chemotaxis chamber at the concentrationindicated along with 1000 ng/ml of HG00606 in both the bottom well andtop part of the filter. Panel A and B show the results obtained with twoindividual donors.

FIGS. 19A and 19B illustrates that HG00606 acts as an antagonist ofEotaxin. Eosinophils were used for chemotaxis assays as described inExample 13. Chemotaxis in response to Eotaxin (closed circles) orEotaxin+HG00606 (closed diamonds) is depicted as the chemotactic indexfrom one representative experiment performed in triplicate. Eotaxin wasadded to the bottom well of the chemotaxis chamber at the concentrationsindicated along with 1000 ng/ml of HG00606 in both the bottom well andtop part of the filter. Panel A and B show the results obtained with twoindividual donors.

FIGS. 20A and 20B demonstrates that HG00606 acts as an antagonist ofCkBeta-10. Eosinophils were used for chemotaxis assays as described inExample 13. Chemotaxis in response to CkBeta-10 (closed circles) orCkBeta-10+HG00606 (closed diamonds) is depicted as the chemotactic indexfrom one representative experiment performed in triplicate. CkBeta-10was added to the bottom well of the chemotaxis chamber at theconcentrations indicated along with 1000 ng/ml of HG00606 in both thebottom well and top part of the filter. Panel A and B show the resultsobtained with two individual donors.

FIG. 21 shows a schematic representation of the pHE4-5 expression vector(SEQ ID NO:21) and the subeloned Ckβ-6 cDNA coding sequence. Thelocations of the kanamycin resistance marker gene, the Ckβ-6 codingsequence, the oriC sequence, and the lacIq coding sequence areindicated.

FIG. 22 shows the nucleotide sequence of the regulatory elements of thepHE promoter (SEQ ID NO:22). The two lac operator sequences, theShine-Delgamo sequence (S/D), and the terminal HindIII and NdeIrestriction sites (italicized) are indicated.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides diagnostic or therapeutic compositionsand methods that utilize isolated polynucleotide molecules encodingCkβ-6 polypeptides, or the Ckβ-6 polypeptides themselves, as well asvectors, host cells and recombinant or synthetic methods for producingsuch compositions. Other names of Ckβ-6 include MPIF-2 and eotoxin-2.

Nucleic Acids

In accordance with an aspect of the present invention, there is providedan isolated nucleic acid (polynucleotide) which encodes for thefull-length or mature polypeptide having the deduced amino acid sequenceof FIG. 1 (SEQ ID NO:2) or for the mature polypeptide encoded by thecDNA of the clone deposited at the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110-2209, as ATCC DepositNo. 75703 on Mar. 10, 1994.

The deposit referred to herein will be maintained under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for purposes of Patent Procedure. The deposit isprovided merely as convenience to those of skill in the art and is notan admission that a deposit is required under 35 U.S.C. §112. Thesequence of the polynucleotide contained in the deposited materials, aswell as the amino acid sequence of the polypeptides encoded thereby, areincorporated herein by reference and are controlling in the event of anyconflict with any description of sequences herein. A license may berequired to make, use or sell the deposited material, and no suchlicense is hereby granted.

The polynucleotide of this invention was discovered from an activatedmonocyte cDNA library. It contains an open reading frame encoding aprotein of approximately 119 amino acids in length of which the first 26amino residues comprise a putative leader sequence. The mature proteintherefore is predicted to be 93 amino acids in length. It isstructurally related to mouse monocyte chemotactic protein-1 (MCP-1 orJE, sequence not shown), and human MCP-1 (SEQ ID NO:5) showing 36%identity, and 52% similarity over the entire human MCP-1 proteinsequence as determined by the computer program Bestfit (shown in FIG.2). The polypeptide contains all four cysteine residues that occur inall chemokines in a characteristic motif. The spacing between thesecysteines is conserved compared with the human MCP-1 and murine MCP-1/JEwhich strongly suggests that the new gene is a chemokine.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in FIG. 1 (SEQ ID NO:1) or that of thedeposited clone or may be a different coding sequence which codingsequence, as a result of the redundancy or degeneracy of the geneticcode, encodes the same mature polypeptide as the DNA of FIG. 1 (SEQ IDNO:1) or the deposited cDNA.

The polynucleotide which encodes for the mature polypeptide of FIG. 1(SEQ ID NO:2) or for the mature polypeptide encoded by the depositedcDNA may include, but is not limited to: only the coding sequence forthe mature polypeptide; the coding sequence for the mature polypeptideand additional coding sequence such as a leader or secretory sequence ora proprotein sequence; the coding sequence for the mature polypeptide(and optionally additional coding sequence) and noncoding sequence, suchas introns or non-coding sequence 5′ and/or 3′ of the coding sequencefor the mature polypeptide.

Thus, the term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc.), and allamino acid sequences of polypeptides encoded by DNA molecules determinedherein were predicted by translation of a DNA sequence determined asabove. Therefore, as is known in the art for any DNA sequence determinedby this automated approach, any nucleotide sequence determined hereinmay contain some errors. Nucleotide sequences determined by automationare typically at least about 90% identical, more typically at leastabout 95% to at least about 99.9% identical to the actual nucleotidesequence of the sequenced DNA molecule. The actual sequence can be moreprecisely determined by other approaches including manual DNA sequencingmethods well known in the art. As is also known in the art, a singleinsertion or deletion in a determined nucleotide sequence compared tothe actual sequence will cause a frame shift in translation of thenucleotide sequence such that the predicted amino acid sequence encodedby a determined nucleotide sequence will be completely different fromthe amino acid sequence actually encoded by the sequenced DNA molecule,beginning at the point of such an insertion or deletion.

Unless otherwise indicated, each nucleotide sequence set forth herein ispresented as a sequence of deoxyribonucleotides (abbreviated A, G, C andT). However, by nucleotide sequence of a nucleic acid molecule orpolynucleotide is intended, for a DNA molecule or polynucleotide, asequence of deoxyribonucleotides, and for an RNA molecule orpolynucleotide, the corresponding sequence of ribonucleotides (A, G, Cand U), where each thymidine deoxyribonucleotide (T) in the specifieddeoxyribonucleotide sequence is replaced by the ribonucleotide uridine(U). For instance, reference to an RNA molecule having the sequence ofSEQ ID NO:1, as set forth using deoxyribonucleotide abbreviations, isintended to indicate an RNA molecule having a sequence in which eachdeoxyribonucleotide A, G or C of SEQ ID NO:1 has been replaced by thecorresponding ribonucleotide A, G or C, and each deoxyribonucleotide Thas been replaced by a ribonucleotide U.

Using the information provided herein, such as the nucleotide sequencein FIG. 1, a nucleic acid molecule of the present invention encoding anCkβ-6 polypeptide may be obtained using standard cloning and screeningprocedures, such as those for cloning cDNAs using mRNA as startingmaterial.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 1 (SEQ ID NO:2) or the polypeptide encoded by the cDNA of thedeposited clone. The variant of the polynucleotide may be a naturallyoccurring allelic variant of the polynucleotide or a non-naturallyoccurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature polypeptide as shown in FIG. 1 (SEQ ID NO:2) or the same maturepolypeptide encoded by the cDNA of the deposited clone as well asvariants of such polynucleotides which variants encode for a fragment,derivative or analog of the polypeptide of FIG. 1 (SEQ ID NO:2) or thepolypeptide encoded by the cDNA of the deposited clone. Such nucleotidevariants include deletion variants, substitution variants and additionor insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIG. 1 (SEQ ID NO:1) or of the coding sequence of the depositedclone. As known in the art, an allelic variant is an alternate form of apolynucleotide sequence which may have a substitution, deletion oraddition of one or more nucleotides, which does not substantially alterthe function of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the codingsequence for the mature polypeptide may be fused in the same readingframe to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5′ amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains.

Thus, for example, the polynucleotide of the present invention mayencode for a mature protein, or for a protein having a prosequence orfor a protein having both a prosequence and a presequence (leadersequence).

The polynucleotides of the present invention can also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence can be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencecan be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell 37:767 (1984)).

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

As indicated, nucleic acid molecules of the present invention may be inthe form of RNA, such as mRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotides orpolypeptides present in a living animal is not isolated, but the samepolynucleotides or DNA or polypeptides, separated from some or all ofthe coexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

Isolated nucleic acid molecules of the present invention include DNAmolecules comprising an open reading frame (ORF) for an Ckβ-6 cDNA; DNAmolecules comprising the coding sequence for a mature Ckβ-6 protein; andDNA molecules which comprise a sequence substantially different fromthose described above but which, due to the degeneracy of the geneticcode, still encode an Ckβ-6 polypeptide. Of course, the genetic code iswell known in the art. Thus, it would be routine for one skilled in theart to generate the degenerate variants described above.

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 70%,preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term“stringent conditions” means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNAs of FIG. 1 (SEQ ID NO:1) orthe deposited cDNA(s).

Alternatively, the polynucleotide may have at least 20 bases, preferably30 bases, and more preferably at least 50 bases which hybridize to apolynucleotide of the present invention and which has an identitythereto, as hereinabove described, and which may or may not retainactivity. For example, such polynucleotides may be employed as probesfor the polynucleotide of SEQ ID NO:1, for example, for recovery of thepolynucleotide or as a diagnostic probe or as a PCR primer.

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide in a nucleicacid molecule of the invention described above, for instance, the cDNAclone contained in ATCC Deposit 75703. By “stringent hybridizationconditions” is intended overnight incubation at 42° C. in a solutioncomprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 g/ml denatured, sheared salmon sperm DNA, followed bywashing the filters in 0.1×SSC at about 65 ° C.

By a polynucleotide which hybridizes to a “portion” of a polynucleotideis intended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt), and more preferably at least about 20 nt,still more preferably at least about 30 nt, and even more preferablyabout 30-70 nt of the reference polynucleotide. These are useful asdiagnostic probes and primers as discussed above and in more detailbelow.

Of course, polynucleotides hybridizing to a larger portion of thereference polynucleotide (e.g. the deposited cDNA clone), for instance,a portion 50-750 nt in length, or even to the entire length of thereference polynucleotide, are also useful as probes according to thepresent invention, as are polynucleotides corresponding to most, if notall, of the nucleotide sequence of the deposited cDNA or the nucleotidesequence as shown in FIG. 1. By a portion of a polynucleotide of “atleast 20 nt in length,” for example, is intended 20 or more contiguousnucleotides from the nucleotide sequence of the referencepolynucleotide. As indicated, such portions are useful diagnosticallyeither as a probe according to conventional DNA hybridization techniquesor as primers for amplification of a target sequence by the polymerasechain reaction (PCR), as described, for instance, in Molecular Cloning,A Laboratory Manual, 2nd. edition, Sambrook, J., Fritsch, E. F. andManiatis, T., eds., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989), the entire disclosure of which is herebyincorporated herein by reference.

Since an Ckβ-6 cDNA clone has been deposited and its determinednucleotide sequence provided, generating polynucleotides which hybridizeto a portion of the Ckβ-6 cDNA molecules would be routine to the skilledartisan. For example, restriction endonuclease cleavage or shearing bysonication of an Ckβ-6 cDNA clone could easily be used to generate DNAportions of various sizes which are polynucleotides that hybridize,respectively, to a portion of the Ckβ-6 cDNA molecule.

Alternatively, the hybridizing polynucleotides of the present inventioncould be generated synthetically according to known techniques. Ofcourse, a polynucleotide which hybridizes only to a poly A sequence(such as the 3. terminal poly(A) tract of a cDNA, or to a complementarystretch of T (or U) residues, would not be included in a polynucleotideof the invention used to hybridize to a portion of a nucleic acid of theinvention, since such a polynucleotide would hybridize to any nucleicacid molecule containing a poly (A) stretch or the complement thereof(e.g. practically any double-stranded cDNA clone).

As indicated, nucleic acid molecules of the present invention whichencode an Ckβ-6 polypeptide may include, but are not limited to thoseencoding the amino acid sequence of the mature polypeptide, by itself;the coding sequence for the mature polypeptide and additional sequences,such as those encoding the leader or secretory sequence, such as a pre-,or pro- or prepro-protein sequence; the coding sequence of the maturepolypeptide, with or without the aforementioned additional codingsequences, together with additional, non-coding sequences, including forexample, but not limited to introns and non-coding 5′ and 3′ sequences,such as the transcribed, non-translated sequences that play a role intranscription, mRNA processing, including splicing and polyadenylationsignals, for example—ribosome binding and stability of mRNA; anadditional coding sequence which codes for additional amino acids, suchas those which provide additional functionalities. Thus, the sequenceencoding the polypeptide may be fused to a marker sequence, such as asequence encoding a peptide which facilitates purification of the fusedpolypeptide. In certain preferred embodiments of this aspect of theinvention, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (Qiagen, Inc.), among others,many of which are commercially available. As described in Gentz, et al.,Proc. Natl. Acad. Sci. (USA) 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. The “HA” tag is another peptide useful for purification whichcorresponds to an epitope derived from the influenza hemagglutininprotein, which has been described by Wilson, et al., Cell 37:767 (1984).As discussed below, other such fusion proteins include at least an Ckβ-6polypeptide or fragment fused to Fc at the N- or C-terminus.

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs orderivatives of an Ckβ-6 polypeptide. Variants may occur naturally, suchas a natural allelic variant. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism. Genes V, Lewin, B., ed., Oxford UniversityPress, New York (1994). Non-naturally occurring variants may be producedusing art-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingregoins, non-coding regions, or both. Alterations in the coding regionsmay produce conservative or non-conservative amino acid substitutions,deletions or additions. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of an Ckβ-6 polypeptide or portions thereof.Also especially preferred in this regard are conservative substitutions.Most highly preferred are nucleic acid molecules encoding the matureprotein or the mature amino acid sequence encoded by the deposited cDNAclone, as described herein.

The present invention is further directed to polynucleotides having atleast a 70% identity, preferably at least 90% and more preferably atleast a 95% identity to a polynucleotide which encodes the polypeptideof SEQ ID NO:2 as well as fragments thereof, which fragments have atleast 30 bases and preferably at least 50 bases and to polypeptidesencoded by such polynucleotides.

Further embodiments of the invention include isolated nucleic acidmolecules comprising a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 96%, 97%, 98% or99% identical to (a) a nucleotide sequence encoding an Ckβ-6 polypeptideor fragment, having an amino acid sequence of FIG. 1 (SEQ ID NO:2),including the predicted leader sequence; (b) a nucleotide sequenceencoding an Ckβ-6 polypeptide having the amino acid sequence of FIG. 1(SEQ ID NO:2), including the predicted leader sequence excepting theN-terminal methionine; (c) a nucleotide sequence encoding the matureCkβ-6 polypeptide (full-length polypeptide with the leader removed); (d)a nucleotide sequence encoding the full-length polypeptide having thecomplete amino acid sequence including the leader encoded by thedeposited cDNA clone; (e) a nucleotide sequence encoding the full-lengthpolypeptide having the complete amino acid sequence including the leaderexcepting the N-terminal methionine encoded by the deposited cDNA clone;(f) a nucleotide sequence encoding the mature polypeptide having theamino acid sequence encoded by the deposited cDNA clone; or (g) anucleotide sequence complementary to any of the nucleotide sequences in(a), (b), (c), (d), (e) or (f).

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding an Ckβ-6polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding thepolypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, thenucleotide sequence shown in FIG. 1, or to the nucleotide sequence ofthe deposited cDNA clone can be determined conventionally using knowncomputer programs such as the Bestfit program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711.Bestfit uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2: 482-489 (1981), to find the bestsegment of homology between two sequences. When using Bestfit or anyother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed.

As one of ordinary skill would appreciate, due to the possibilities ofsequencing errors discussed above, as well as the variability ofcleavage sites for leaders in different known proteins, the actualmature Ckβ-6 polypeptide encoded by the deposited cDNA comprises about93 amino acids, but may be anywhere in the range of 86-99 amino acids;and the actual leader sequence of this protein is about 26 amino acids,but may be anywhere in the range of about 20 to about 33 amino acids.

Such isolated molecules, particularly DNA molecules, are useful asprobes for gene mapping, by in situ hybridization with chromosomes, andfor detecting expression of an Ckβ-6 gene in human tissue, for instance,by Northern blot analysis. The present invention is further directed tofragments of the isolated nucleic acid molecules described herein. By afragment of an isolated nucleic acid molecule having the nucleotidesequence of the deposited Ckβ-6 cDNA, or a nucleotide sequence shown inFIG. 1 (SEQ ID NO:1), is intended fragments at least about 15 nt, andmore preferably at least about 20 nt, still more preferably at leastabout 30 nt, and even more preferably, at least about 40 nt in lengthwhich are useful as diagnostic probes and primers as discussed herein.Of course, larger fragments 50-500 nt in length are also usefulaccording to the present invention as are fragments corresponding tomost, if not all, of a nucleotide sequence of the deposited Ckβ-6 cDNAs,or as shown in FIG. 1 (SEQ ID NO:1). By a fragment at least 20 nt inlength, for example, is intended fragments which include 20 or morecontiguous bases from the nucleotide sequence of the deposited cDNA orthe nucleotide sequence as shown in FIG. 1 (SEQ ID NO:1). Since the genehas been deposited and the nucleotide sequences shown in FIG. 1 (SEQ IDNO:1) is provided, generating such DNA fragments would be routine to theskilled artisan. For example, restriction endonuclease cleavage orshearing by sonication could easily be used to generate fragments ofvarious sizes. Alternatively, such fragments could be generatedsynthetically.

Fragments of the full length gene of the present invention may be usedas a hybridization probe for a cDNA library to isolate the full lengthcDNA and to isolate other cDNAs which have a high sequence similarity tothe gene or similar biological activity. Probes of this type preferablyhave at least 30 bases and may contain, for example, 50 or more bases,The probe may also be used to identify a cDNA clone corresponding to afull length transcript and a genomic clone or clones that contain thecomplete gene including regulatory and promotor regions, exons, andintrons. An example of a screen comprises isolating the coding region ofthe gene by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

Polypeptides and Polypeptide Fragments

The present invention further relates to an isolated polypeptide whichhas the deduced amino acid sequence of FIG. 1 (SEQ ID NO:2) or which hasthe amino acid sequence encoded by the deposited cDNA, as well asfragments, analogs and derivatives of such polypeptide. The terms“peptide” and “oligopeptide” are considered synonymous (as is commonlyrecognized) and each term can be used interchangeably as the contextrequires to indicate a chain of at least two amino acids coupled bypeptidyl linkages. The word “polypeptide” is used herein for chainscontaining more than ten amino acid residues. All oligopeptide andpolypeptide formulas or sequences herein are written from left to rightand in the direction from amino terminus to carboxy terminus.

By “a polypeptide having Ckβ-6 activity” is intended polypeptidesexhibiting activity similar, but not necessarily identical, to anactivity of the Ckβ-6 protein of the invention (either the full-lengthprotein or, preferably, the mature protein), as measured in a particularbiological assay. Ckβ-6 protein activity can be measured by the assaysset forth in Examples 5, 6, 7 and 8. For example, Ckβ-6 protein activitymeasured using the in vitro myeloprotection assay disclosed in Example7, infra.

Briefly, lineagedepleted populations of cells (Lin⁻ cells) are isolatedfrom mouse bone marrow and incubated in the presence of multiplecytokines with or without Ckβ-6. After 48 hours, one set of each culturereceives 5-Fu and the incubation is then continued for additional 24hours, at which point the numbers of surviving low proliferativepotential colony-forming cells (LPP-CFC) and high proliferativepotential colony-forming cells (HPP-CFC) are determined by any suitableclonogenic assay known to those of skill in the art.

Thus, “a polypeptide having Ckβ-6 protein activity” includespolypeptides that exhibit Ckβ-6 activity, in the above-described assay.Although the degree of activity need not be identical to that of theCkβ-6 protein, preferably, “a polypeptide having Ckβ-6 protein activity”will exhibit substantially similar activity as compared to the Ckβ-6protein (i.e., the candidate polypeptide will exhibit greater activityor not more than about twenty-fold less and, preferably, not more thanabout ten-fold less activity relative to the reference Ckβ-6 protein).

The present invention further relates to Ckβ-6 polypeptides which havethe amino acid sequence of FIG. 1 (SEQ ID NO:2) or which have the aminoacid sequence encoded by the deposited cDNA, as well as fragments,analogs and derivatives of such polypeptide.

The terms “fragment,” “derivative” and “analog” when referring to thepolypeptide of FIG. 1 (SEQ ID NO:2) or that encoded by the depositedcDNA, means a polypeptide which retains essentially the same biologicalfunction or activity as such polypeptide. Thus, an analog includes aproprotein which can be activated by cleavage of the proprotein portionto produce an active mature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 1 (SEQ IDNO:2) or that encoded by the deposited cDNA may be (i) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The polypeptides of the present invention include the polypeptide of SEQID NO:2 (in particular the mature polypeptide) as well as polypeptideswhich have at least 70% similarity (preferably at least 70% identity) tothe polypeptide of SEQ ID NO:2 and more preferably at least 90%similarity (more preferably at least 90% identity) to the polypeptide ofSEQ ID NO:2 and still more preferably at least 95% similarity (stillmore preferably at least 95% identity) to the polypeptide of SEQ ID NO:2and also include portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%,98%, or 99% identical to the nucleic acid sequence of the deposited cDNA(ATCC 75703) or the nucleic acid sequence shown in FIG. 1 (SEQ ID NO:1)will encode a polypeptide “having Ckβ-6 protein activity”. In fact,since degenerate variants of these nucleotide sequences all encode thesame polypeptide, this will be clear to the skilled artisan even withoutperforming the above described comparison assay. It will be furtherrecognized in the art that, for such nucleic acid molecules that are notdegenerate variants, a reasonable number will also encode a polypeptidehaving Ckβ-6 protein activity. This is because the skilled artisan isfully aware of amino acid substitutions that are either less likely ornot likely to significantly effect protein function (e.g. replacing onealiphatic amino acid with a second aliphatic amino acid).

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie, J. U., et al., “Deciphering theMessage in Protein Sequences: Tolerance to Amino Acid Substitutions,”Science 247:1306-1310 (1990), wherein the authors indicate that thereare two main approaches for studying the tolerance of an amino acidsequence to change. The first method relies on the process of evolution,in which mutations are either accepted or rejected by natural selection.The second approach uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene and selections or screensto identify sequences that maintain functionality. As the authors state,these studies have revealed that proteins are surprisingly tolerant ofamino acid substitutions. The authors further indicate which amino acidchanges are likely to be permissive at a certain position of theprotein. For example, most buried amino acid residues require nonpolarside chains, whereas few features of surface side chains are generallyconserved. Other such phenotypically silent substitutions are describedin Bowie, J. U. et al., supra, and the references cited therein.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

In addition, polypeptides of the invention may also include an initialmodified methionine residue, in some cases as a result of host-mediatedprocesses. Thus, it is well known in the art that the N-terminalmethionine encoded by the translation initiation codon generally isremoved with high efficiency from any protein after translation in alleukaryotic cells. While the N-terminal methionine on most proteins alsois efficiently removed in most prokaryotes, for some proteins thisprokaryotic removal process is inefficient, depending on the nature ofthe amino acid to which the N-terminal methionine is covalently linked.Thus, a full-length Ckβ-6 polypeptide lacking the N-terminal methionineis specifically contemplated by the invention. Further, it will berecognized by those of skill in the art that in many cases it may bebeneficial to add an N-terminal methionine to an N-terminally truncatedCkβ-6 polypeptide otherwise lacking an amino terminal methionine, forexample, to achieve efficient expression by recombinant technology inbacterial such as E. coli.

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals, but also additionalheterologous functional regions. For instance, a region of additionalamino acids, particularly charged amino acids, may be added to theN-terminus of the polypeptide to improve stability and persistence inthe host cell, during purification, or during subsequent handling andstorage. Also, peptide moieties may be added to the polypeptide tofacilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilizeproteins. For example, EP-A-O 464 533 (Canadian counterpart 2045869)discloses fusion proteins comprising various portions of constant regionof immunoglobin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is thoroughlyadvantageous for use in therapy and diagnosis and thus results, forexample, in improved pharmacokinetic properties (EP-A 0232 262). On theother hand, for some uses it would be desirable to be able to delete theFc part after the fusion protein has been expressed, detected andpurified in the advantageous manner described. This is the case when Fcportion proves to be a hindrance to use in therapy and diagnosis, forexample when the fusion protein is to be used as antigen forimmunizations. In drug discovery, for example, human proteins, such as,hIL5- has been fused with Fc portions for the purpose of high-throughputscreening assays to identify antagonists of hIL-5. See, Bennett, D., etal., Journal of Molecular Recognition 8:52-58 (1995) and Johanson, K.,et al., J. Biol. Chem. 270(16):9459-9471 (1995).

The Ckβ-6 protein can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification. Polypeptides of the presentinvention include naturally purified products, products of chemicalsynthetic procedures, and products produced by recombinant techniquesfrom a prokaryotic or eukaryotic host, including, for example,bacterial, yeast, higher plant, insect and mammalian cells. Dependingupon the host employed in a recombinant production procedure, thepolypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes.

It will be recognized in the art that some amino acid sequences of theCkβ-6 polypeptide can be varied without significant affect on thestructure or function of the protein. If such differences in sequenceare contemplated, it should be remembered that there will be criticalareas on the protein which determine activity. In general, it ispossible to replace residues which form the tertiary structure, providedthat residues performing a similar function are used. In otherinstances, the type of residue may be completely unimportant if thealteration occurs at a non-critical region of the protein.

Thus, the invention further includes variations of an Ckβ-6 polypeptidewhich show substantial Ckβ-6 polypeptide activity or which includeregions of an Ckβ-6 protein such as the protein portions discussedbelow. Such mutants include deletions, insertions, inversions, repeats,and type substitutions (for example, substituting one hydrophilicresidue for another, but not strongly hydrophilic for stronglyhydrophobic as a rule). Small changes or such “neutral” amino acidsubstitutions will generally have little effect on activity.

Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu and Ile;interchange of the hydroxyl residues Ser and Thr, exchange of the acidicresidues Asp and Glu, substitution between the amide residues Asn andGln, exchange of the basic residues Lys and Arg and replacements amongthe aromatic residues Phe, Tyr.

Of additional special interest are also substitutions of charged aminoacids with another charged amino acid or with neutral amino acids. Thismay result in proteins with improved characteristics such as lessaggregation. Prevention of aggregation is highly desirable. Aggregationof proteins cannot only result in a reduced activity but be problematicwhen preparing pharmaceutical formulations because they can beimmunogenic (Pinckard, et al., Clin. Exp. Immunol. 2:331-340 (1967),Robbins, et al., Diabetes 36:838-845 (1987), Cleland, et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).

The replacement of amino acids can also change the selectivity of thebinding to cell surface receptors. Ostade, et al., Nature 361:266-268(1993), described certain TNF alpha mutations resulting in selectivebinding of TNF alpha to only one of the two known TNF receptors.

As indicated in detail above, further guidance concerning which aminoacid changes are likely to be phenotypically silent (i.e., are notlikely to have a significant deleterious effect on a function) can befound in Bowie, J. U., et al., “Deciphering the Message in ProteinSequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310(1990) (see Table 1).

As indicated, changes are preferably of a minor nature, such asconservative amino acid-substitutions that do not significantly affectthe folding or activity of the protein (see Table 1).

TABLE 1 Conservative Amino Acid Substitutions Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

Of course, the number of amino acid substitutions a skilled artisanwould make depends on many factors, including those described above.Generally speaking, the number of substitutions for any given Ckβ-6polypeptide will not bemorethan50, 40, 30, 25, 20, 15, 10, 5or 3.

Recombinant DNA technology known to those skilled in the art can be usedto create novel proteins. Muteins and deletions or fusion proteins canshow, e.g., enhanced activity or increased stability. In addition, theycould be purified in higher yields and show better solubility at leastunder certain purification and storage conditions. Set out below areadditional examples of mutations that can be constructed.

Ckβ-6 Amino terminal and carboxy terminal deletions: Interferon gammashows up to ten times higher activities by deleting 8-10 amino acidresidues from the carboxy terminus of the protein (D{haeck over(s)}beli, et al., J. Biotechnology 7:199-216 (1988)). Ron, et al., J.Biol. Chem. 268(4):2984-2988 (1993), reported modified KGF proteins thathad heparin binding activity even if 3, 8, or 27 amino terminal aminoacid residues were missing. Many other examples are known to thoseskilled in the art.

In the present case it has been shown that the Ckβ-6 polypeptidecomposition, comprising C-termiinal Ckβ-6 truncations, producedaccording to the method of Example 3 retains Ckβ-6 polypeptide activity.Furthermore, since the protein of the invention is a member of thechemokine polypeptide family, deletions of N-terminal amino acids up tothe cysteine at position 7 of SEQ ID NO:2 (Cys-7), and deletions ofC-terminal amino acids up to the cysteine at position 48 of SEQ ID NO:2(Cys-48), may retain some biological activity such as receptor bindingor modulation of target cell activities. Polypeptides having further N-and C-terminal deletions including Cys-7 or Cys-48 would not be expectedto retain such biological activities because it is known that theseresidues in a chemokine-related polypeptide are required for forming adisulfide bridge to provide structural stability which is needed forreceptor binding and signal transduction.

However, even if deletion of one or more amino acids from the N- orC-termini of a protein results in modification of loss of one or morebiological functions of the protein, other biological activities maystill be retained. Thus, the ability of the shortened protein to induceand/or bind to antibodies which recognize the complete or mature form ofthe protein generally will be retained when less than the majority ofthe residues of the complete or mature protein are removed from eithertermini. Whether a particular polypeptide lacking N- or C-terminalresidues of a complete protein retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the amino acidsequence of the Ckβ-6 polypeptide shown in SEQ ID NO:2 up to thecysteine as position 7. Likewise the present invention providespolypeptides having one or more residues deleted from the carboxyterminus of the amino acid sequence of the Ckβ-6 polypeptide shown inSEQ ID NO:2, up to the cysteine at position 48. In particular, thepresent invention provides polypeptides having the amino acid sequenceof residues n-93 of the amino acid sequence in SEQ ID NO:2, where n isany integer in the range of 1-7. Similarly, the present inventionprovides polypeptides having the amino acid sequence of residues 1-m ofthe amino acid sequence in SEQ ID NO:2, where m is any integer in therange of 48-93.

The invention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini, which may bedescribed generally as having residues x-n-m of SEQ ID NO:2, where n andm are integers as described above, and x may be either NH₂ ormethionine.

More in particular, the invention provides polypeptides having the aminoacid sequence of residues:

Val (1) to Cys (93) Pro (4) to Cys (93) Val (2) to Cys (93) Ser (5) toCys (93) Ile (3) to Cys (93) Pro (6) to Cys (93) Val (1) to Thr (92) Pro(4) to Thr (92) Val (2) to Thr (92) Ser (5) to Thr (92) Ile (3) to Thr(92) Pro (6) to Thr (92) Val (1) to Thr (91) Pro (4) to Thr (91) Val (2)to Thr (91) Ser (5) to Thr (91) Ile (3) to Thr (91) Pro (6) to Thr (91)Val (I) to Gln (90) Pro (4) to Gln (90) Val (2) to Gln (90) Ser (5) toGln (90) Ile (3) to Gln (90) Pro (6) to Gln (90) Val (1) to Asn (89) Pro(4) to Asn (89) Val (2) to Asn (89) Ser (5) to Asn (89) Ile (3) to Asn(89) Pro (6) to Asn (89) Val (1) to Gly (88) Pro (4) to Gly (88) Val (2)to Gly (88) Ser (5) to Gly (88) Ile (3) to Gly (88) Pro (6) to Gly (88)Val (1) to Pro (87) Pro (4) to Pro (87) Val (2) to Pro (87) Ser (5) toPro (87) Ile (3) to Pro (87) Pro (6) to Pro (87) Val (l) to Tyr (86) Pro(4) to Tyr (86) Val (2) to Tyr (86) Ser (5) to Tyr (86) Ile (3) to Tyr(86) Pro (6) to Tyr (86) Val (1) to Arg (85) Pro (4) to Arg (85) Val (2)to Arg (85) Ser (5) to Arg (85) Ile (3) to Arg (85) Pro (6) to Arg (85)Val (1) to Gln (84) Pro (4) to Gln (84) Val (2) to Gln (84) Ser (5) toGln (84) Ile (3) to Gln (84) Pro (6) to Gln (84) Val (1) to Val (83) Pro(4) to Val (83) Val (2) to Val (83) Ser (5) to Val (83) Ile (3) to Val(83) Pro (6) to Val (83) Val (1) to Pro (82) Pro (4) to Pro (82) Val (2)to Pro (82) Ser (5) to Pro (82) Ile (3) to Pro (82) Pro (6) to Pro (82)Val (1) to Gly (81) Pro (4) to Gly (81) Val (2) to Gly (81) Ser (5) toGly (81) Ile (3) to Gly (81) Pro (6) to Gly (81) Val (1) to Lys (80) Pro(4) to Lys (80) Val (2) to Lys (80) Ser (5) to Lys (80) Ile (3) to Lys(80) Pro (6) to Lys (80) Val (1) to Val (79) Pro (4) to Val (79) Val (2)to Val (79) Ser (5) to Val (79) Ile (3) to Val (79) Pro (6) to Val (79)Val (1) to Ala (78) Pro (4) to Ala (78) Val (2) to Ala (78) Ser (5) toAla (78) Ile (3) to Ala (78) Pro (6) to Ala (78) Val (1) to Val (77) Pro(4) to Val (77) Val (2) to Val (77) Ser (5) to Val (77) Ile (3) to Val(77) Pro (6) to Val (77) Val (1) to Ala (76) Pro (4) to Ala (76) Val (2)to Ala (76) Ser (5) to Ala (76) Ile (3) to Ala (76) Pro (6) to Ala (76)Val (1) to Arg (75) Pro (4) to Arg (75) Val (2) to Arg (75) Ser (5) toArg (75) Ile (3) to Arg (75) Pro (6) to Arg (75) Val (1) to Ala (74) Pro(4) to Ala (74) Val (2) to Ala (74) Ser (5) to Ala (74) Ile (3) to Ala(74) Pro (6) to Ala (74) Val (1) to Arg (73) Pro (4) to Arg (73) Val (2)to Arg (73) Ser (5) to Arg (73) Ile (3) to Arg (73) Pro (6) to Arg (73)Val (1) to Pro (72) Pro (4) to Pro (72) Val (2) to Pro (72) Ser (5) toPro (72) Ile (3) to Pro (72) Pro (6) to Pro (72) Val (1) to Ser (71) Pro(4) to Ser (71) Val (2) to Ser (71) Ser (5) to Ser (71) Ile (3) to Ser(71) Pro (6) to Ser (71) Val (1) to Ala (70) Pro (4) to Ala (70) Val (2)to Ala (70) Ser (5) to Ala (70) Ile (3) to Ala (70) Pro (6) to Ala (70)Val (1) to Lys (69) Pro (4) to Lys (69) Val (2) to Lys (69) Ser (5) toLys (69) Ile (3) to Lys (69) Pro (6) to Lys (69) Val (1) to Lys (68) Pro(4) to Lys (68) Val (2) to Lys (68) Ser (5) to Lys (68) Ile (3) to Lys(68) Pro (6) to Lys (68) Val (1) to Gln (67) Pro (4) to Gln (67) Val (2)to Gln (67) Ser (5) to Gla (67) Ile (3) to Gln (67) Pro (6) to Gln (67)Val (1) to Lys (66) Pro (4) to Lys (66) Val (2) to Lys (66) Ser (5) toLys (66) Ile (3) to Lys (66) Pro (6) to Lys (66) Val (1) to Ala (65) Pro(4) to Ala (65) Val (2) to Ala (65) Pro (5) to Ala (65) Ile (3) to Ala(65) Pro (6) to Ala (65) Val (1) to Asp (64) Pro (4) to Asp (64) Val (2)to Asp (64) Ser (5) to Asp (64) Ile (3) to Asp (64) Pro (6) to Asp (64)Val (1) to Leu (63) Pro (4) to Leu (63) Val (2) to Leu (63) Ser (5) toLeu (63) Ile (3) to Leu (63) Pro (6) to Leu (63) Val (1) to Asn (62) Pro(4) to Asn (62) Val (2) to Asn (62) Ser (5) to Asn (62) Ile (3) to Asn(62) Pro (6) to Asn (62) Val (1) to Lys (61) Pro (4) to Lys (61) Val (2)to Lys (61) Ser (5) to Lys (61) Ile (3) to Lys (61) Pro (6) to Lys (61)Val (1) to Met (60) Pro (4) to Met (60) Val (2) to Met (60) Ser (5) toMet (60) Ile (3) to Met (60) Pro (6) to Met (60) Val (1) to Tyr (59) Pro(4) to Tyr (59) Val (2) to Tyr (59) Ser (5) to Tyr (59) Ile (3) to Tyr(59) Pro (6) to Tyr (59) Val (1) to Arg (58) Pro (4) to Arg (58) Val (2)to Arg (58) Ser (5) to Arg (58) Ile (3) to Arg (58) Pro (6) to Arg (58)Val (1) to Gln (57) Pro (4) to Gln (57) Val (2) to Gln (57) Ser (5) toGln (57) Ile (3) to Gln (57) Pro (6) to Gln (57) Val (1) to Val (56) Pro(4) to Val (56) Val (2) to Val (56) Ser (5) to Val (56) Ile (3) to Val(56) Pro (6) to Val (56) Val (1) to Trp (55) Pro (4) to Trp (55) Val (2)to Trp (55) Ser (5) to Trp (55) Ile (3) to Trp (55) Pro (6) to Trp (55)Val (1) to Glu (54) Pro (4) to Glu (54) Val (2) to Glu (54) Ser (5) toGlu (54) Ile (3) to Glu (54) Pro (6) to Glu (54) Val (1) to Gln (53) Pro(4) to Gln (53) Val (2) to Gln (53) Ser (5) to Gln (53) Ile (3) to Gln(53) Pro (6) to Gln (53) Val (1) to Lys (52) Pro (4) to Lys (52) Val (2)to Lys (52) Ser (5) to Lys (52) Ile (3) to Lys (52) Pro (6) to Lys (52)Val (1) to Pro (51) Pro (4) to Pro (51) Val (2) to Pro (51) Ser (5) toPro (51) Ile (3) to Pro (51) Pro (6) to Pro (51) Val (1) to Asp (50) Pro(4) to Asp (50) Val (2) to Asp (50) Ser (5) to Asp (50) Ile (3) to Asp(50) Pro (6) to Asp (50) Val (1) to Gly (49) Pro (4) to Gly (49) Val (2)to Gly (49) Ser (5) to Gly (49) Ile (3) to Gly (49) Pro (6) to Gly (49)Val (1) to Cys (48) Pro (4) to Cys (48) Val (2) to Cys (48) Ser (5) toCys (48) Ile (3) to Cys (48) Pro (6) to Cys (48)

all of SEQ ID NO:2. The polypeptides described above may also have anN-terminal methionine. Polynucleotides encoding these polypeptides alsoare provided. Also included are a nucleotide sequence encoding apolypeptide consisting of a portion of the complete Ckβ-6 amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 75703,where this portion excludes from 1 to about 6 amino acids from the aminoterminus of the mature amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No. 75703, or from 1 to about 45 amino acidsfrom the carboxy terminus, or any combination of the above aminoterminal and carboxy terminal deletions, of the complete amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 75703.Polynucleotides encoding all of the above deletion mutant polypeptideforms also are provided.

Particularly preferred Ckβ-6 C-terminal truncations are shown below(numbering is as shown in SEQ ID NO:2):

Val(1) to Ala (78);

Val (1) to Ala (76);

Val (1) to Arg (75); and

Val (1) to Arg (73).

Substitution of amino acids: A further aspect of the present inventionalso includes the substitution of amino acids. Of special interest areconservative amino acid substitutions at do not significantly affect thefolding of the protein. Examples of conservative amino acidsubstitutions known to those skilled in the art are set forth Table 1,above.

Of additional special interest are also substitutions of charged aminoacids with another charged amino acid or with neutral amino acids. Thismay result in proteins with improved characteristics such as lessaggregation. Prevention of aggregation is highly desirable. Aggregationof proteins cannot only result in a reduced activity but be problematicwhen preparing pharmaceutical formulations because they can beimmunogenic (Pinckard, et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins, et al., Diabetes 36:838-845 (1987); Cleland, et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993)).

The Ckβ-6 protein may contain one or several amino acid substitutions,deletions or additions, either from natural mutation or humanmanipulation. Examples of some preferred mutations are:

Lys (42) to Ser

Lys (43) to Ser

The lysine residues at positions 42 and 43 of SEQ ID NO:2 correspondwith a site known to be necessary for heparin binding in otherchemokines. These substitutions would be expected to generate Ckβ-6antagonists by destroying the ability of Ckβ-6 to bind heparin (Graham,G. J. et al., EMBO 15:6506-15 (1996)).

Asp (50) to Ala, Gly, Ser, Thr or Met,

Asp (64) to Ala, Gly, Ser, Thr or Met,

Substitutions of Asp-50 and Asp-64 of SEQ ID NO:2 have been predicted bythe inventors herein to increase Ckβ-6 polypeptide activity by improvingthe dimerization potential of such polypeptides.

Phe (47) to Ser

The polypeptide composition generated in Example 3 is thought to containa mutation of the codon -TTC- which codes for Phe47 in SEQ ID NO:2. Suchmutation has resulted in the codon -TCC- which codes for Ser. Thismutation is thought to have been generated by Taq DNA polymerase duringthe polymerase chain reaction used during the subeloning of the Ckβ-6cDNA into the expression vector as described in Example 3. Taqpolymerase is known by those of skill in the art to possess less thanperfect fidelity.

The present invention further provides for a Ckβ-6 agonist polypeptideswherein the amino terminus of said polypeptides is a residue selectedfrom residue 2 or residue 3 of SEQ ID NO:2 and the carboxy terminus ofsaid polypeptides is residue m, wherein m is any residue from residue 48to residue 93 of SEQ ID NO:2. Specific Ckβ-6 agonists according to thepresent invention include: Val(1) to Ala(78); Val(1) to Val(77); Val(1)to Ala(76); Val(1) to Arg(75); Val(1) to Arg(73); Val(2) to Ala(78);Val(2) to Val(77); Val(2) to Ala(76); Val(2) to Arg(75); Val(2) toArg(73); Ile(3) to Ala(78); Ile(3) to Val (77); Ile(3) to Ala (76);Ile(3) to Arg(75); Ile(3) to Arg(73). The agonist of the presentinvention may have either NH2 or methionine attached to the N-terminus.

The present invention further relates to Ckβ-6 antagonists. Inparticular, a deletion of the first three N-terminal amino acid residuesof the mature CW4B-6 protein (i.e., a deletion of Val(1) to Ile(3) inSEQ ID NO:2) results in a polypeptide having antagonistic activity.Thus, according to the present invention, Ckβ-6 antagonists are providedwherein the amino terminus is residue 4 of SEQ ID NO:2 and the carboxylterminus is residue m, wherein m is any residue of SEQ ID NO:2 fromresidue 48 to residue 93. Specific Ckβ-6 antagonists according to thepresent invention include, but are not limited to: Pro(4) to Arg(73);Pro(4) to Arg(75); Pro(4) to Ala(76); Pro(4) to Ala(78). Optionally, theCkβ-6 antagonists of the present invention can include a Met residue atthe N-terminus.

It has been discovered that the present Ckβ-6 antagonist inhibits notonly the activity of Ckβ-6 and agonists of Ckβ-6, but also the activityof other chemokines, such as Ckβ-10 and Eotaxin (See Example 13 belowwhere a Ckβ-6 antagonist polypeptide of the present invention was shownto inhibit eosinophil chemotaxis driven by Ckβ-6, Ckβ-10, or Eotaxin.Ckβ-6, Ckβ-10, and Eotaxin all mediate their effects on eosinophils viathe CCR3 receptor. Thus, the antagonist polypeptides of the presentinvention represent dominant antagonists which are capable of inhibitingCCR3 receptor signaling regardless of the chemokine mediating thiseffect. Accordingly, by the invention, a method is provided forinhibiting the CCR3 receptor signaling pathway comprising administeringto cells which express the CCR3 receptor an effective amount of a Ckβ-6antagonist of the present invention. An “effective amount” of a Ckβ-6antagonist and several disease conditions due to the activation ofeosinophils are discussed below.

It will be appreciated by those of skill in the art that Ckβ-6polypeptides, including those Ckβ-6 polypeptides having N- andC-terminal deletions, can contain one or more of the abovesubstitutions.

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Arecombinantly produced version of the Ckβ-6 polypeptide can besubstantially purified by the one-step method described in Smith andJohnson, Gene 67:3140 (1988).

The polypeptides of the present invention include the polypeptideencoded by the deposited cDNA including the leader, the polypeptideencoded by the deposited cDNA including the leader excepting theN-terminal methionine, the mature polypeptide encoded by the depositedcDNA minus the leader (i.e., the mature protein), the polypeptide ofFIG. 1 (SEQ ID NO:2) including the leader, the polypeptide of FIG. 1(SEQ ID NO:2) including the leader excepting the N-terminal methionine,the polypeptide of FIG. 1 (SEQ ID NO:2) minus the leader, as well aspolypeptides which have at least 90% similarity, more preferably atleast 95% similarity, and still more preferably at least 96%, 97%, 98%or 99% similarity to those described above. Further polypeptides of thepresent invention include polypeptides at least 80% identical, morepreferably at least 90% or 95% identical, still more preferably at least96%, 97%, 98% or 99% identical to the polypeptide encoded by thedeposited cDNA, to the polypeptide of FIG. 1 (SEQ ID NO:2) and alsoinclude portions of such polypeptides with at least 30 amino acids andmore preferably at least 50 amino acids.

By “% similarity” for two polypeptides is intended a similarity scoreproduced by comparing the amino acid sequences of the two polypeptidesusing the Bestfit program (Wisconsin Sequence Analysis Package, Version8 for Unix, Genetics Computer Group, University Research Park, 575Science Drive, Madison, Wis. 53711) and the default settings fordetermining similarity. Bestfit uses the local homology algorithm ofSmith and Waterman (Advances in Applied Mathematics 2:482-489, 1981) tofind the best segment of similarity between two sequences.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of an Ckβ-6polypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of the Ckβ-6 polypeptide. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular polypeptide is at least90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequence shown in FIG. 1 (SEQ ID NO:2) or to the amino acidsequence encoded by the deposited cDNA clone can be determinedconventionally using known computer programs such the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711. When using Bestfit or any other sequence alignment programto determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set, of course, such that the percentage of identityis calculated over the full length of the reference amino acid sequenceand that gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed.

The polypeptide of the present invention could be used as a molecularweight marker on SDS-PAGE gels or on molecular sieve gel filtrationcolumns using methods well known to those of skill in the art.

As described in detail below, the polypeptides of the present inventioncan also be used to raise polyclonal and monoclonal antibodies, whichare useful in assays for detecting Ckβ-6 protein expression as describedbelow or as agonists and antagonists capable of enhancing or inhibitingCkβ-6 protein function. Further, such polypeptides can be used in theyeast two-hybrid system to “capture” Ckβ-6 protein binding proteinswhich are also candidate agonist and antagonist according to the presentinvention. The yeast two hybrid system is described in Fields and Song,Nature 340:245-246 (1989).

In another aspect, the invention provides a peptide or polypeptidecomprising an epitope-bearing portion of a polypeptide of the invention.The epitope of this polypeptide portion is an immunogenic or antigenicepitope of a polypeptide of the invention. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response whenthe whole protein is the immunogen. These immunogenic epitopes arebelieved to be confined to a few loci on the molecule. On the otherhand, a region of a protein molecule to which an antibody can bind isdefined as an “antigenic epitope.” The number of immunogenic epitopes ofa protein generally is less than the number of antigenic epitopes. See,for instance, Geysen, et al., Proc. Natl. Acad. Sci. USA 81:3998-4002(1983).

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, e.g., Sutcliffe, J. G., et al., Science219:660-666 (1983).

Peptides capable of eliciting protein-reactive sera are frequentlyrepresented in the primary sequence of a protein, can be characterizedby a set of simple chemical rules, and are confined neither toimmunodominant regions of intact proteins (i.e., immunogenic epitopes)nor to the amino or carboxyl terminals. Peptides that are extremelyhydrophobic and those of six or fewer residues generally are ineffectiveat inducing antibodies that bind to the mimicked protein; longer,peptides, especially those containing proline residues, usually areeffective. Sutcliffe et al., supra, at 661. For instance, 18 of 20peptides designed according to these guidelines, containing 8-39residues covering 75% of the sequence of the influenza virushemagglutinin HA1 polypeptide chain, induced antibodies that reactedwith the HA1 protein or intact virus; and 12/12 peptides from the MULVpolymerase and 18/18 from the rabies glycoprotein induced antibodiesthat precipitated the respective proteins.

Antigenic epitope-bearing peptides and polypeptides of the invention aretherefore useful to raise antibodies, including monoclonal antibodies,that bind specifically to a polypeptide of the invention. Thus, a highproportion of hybridomas obtained by fusion of spleen cells from donorsimmunized with an antigen epitope-bearing peptide generally secreteantibody reactive with the native protein. Sutcliffe et al., supra, at663. The antibodies raised by antigenic epitope-bearing peptides orpolypeptides are useful to detect the mimicked protein, and antibodiesto different peptides may be used for tracking the fate of variousregions of a protein precursor which undergoes post-translationalprocessing. The peptides and anti-peptide antibodies may be used in avariety of qualitative or quantitative assays for the mimicked protein,for instance in competition assays since it has been shown that evenshort peptides (e.g. about 9 amino acids) can bind and displace thelarger peptides in immunoprecipitation assays. See, for instance,Wilson, et al., Cell 37:767-778 (1984) at 777. The anti-peptideantibodies of the invention also are useful for purification of themimicked protein, for instance, by adsorption chromatography usingmethods well known in the art.

Antigenic epitope-bearing peptides and polypeptides of the inventiondesigned according to the above guidelines preferably contain a sequenceof at least seven, more preferably at least nine and most preferablybetween about 15 to about 30 amino acids contained within the amino acidsequence of a polypeptide of the invention. However, peptides orpolypeptides comprising a larger portion of an amino acid sequence of apolypeptide of the invention, containing about 30 to about 50 aminoacids, or any length up to and including the entire amino acid sequenceof a polypeptide of the invention, also are considered epitope-bearingpeptides or polypeptides of the invention and also are useful forinducing antibodies that react with the mimicked protein. Preferably,the amino acid sequence of the epitope-bearing peptide is selected toprovide substantial solubility in aqueous solvents (i.e., the sequenceincludes relatively hydrophilic residues and highly hydrophobicsequences are preferably avoided); and sequences containing prolineresidues are particularly preferred.

Non-limiting examples of antigenic polypeptides or peptides that can beused to generate Ckβ-6-specific antibodies include: a polypeptidecomprising amino acid residues from about Val-12 to about Val-21 in FIG.1 (SEQ ID NO:2); a polypeptide comprising amino acid residues from aboutLeu-26 to about Lys-34 in FIG. 1 (SEQ ID NO:2); a polypeptide comprisingamino acid residues from about Phe-39 to about Cys-48 in FIG. 1 (SEQ IDNO:2); and a polypeptide comprising amino acid residues from aboutAsp-50 to about Gln-57 in FIG. 1 (SEQ ID NO:2). As indicated above, theinventors have determined that the above polypeptide fragments areantigenic regions of the Ckβ-6 protein.

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means for making peptides or polypeptidesincluding recombinant means using nucleic acid molecules of theinvention. For instance, a short epitope-bearing amino acid sequence maybe fused to a larger polypeptide which acts as a carrier duringrecombinant production and purification, as well as during immunizationto produce anti-peptide antibodies. Epitope-bearing peptides also may besynthesized using known methods of chemical synthesis. For instance,Houghten has described a simple method for synthesis of large numbers ofpeptides, such as 10-20 mg of 248 different 13 residue peptidesrepresenting single amino acid variants of a segment of the HA1polypeptide which were prepared and characterized (by ELISA-type bindingstudies) in less than four weeks. Houghten, R. A., “General method forthe rapid solid-phase synthesis of large numbers of peptides:specificity of antigen-antibody interaction at the level of individualamino acids,” Proc. Natl. Acad. Sci. (USA) 82:5131-5135 (1985). This“Simultaneous Multiple Peptide Synthesis (SMPS)” process is furtherdescribed in U.S. Pat. No. 4,631,211 to Houghten, et al. (1986). In thisprocedure the individual resins for the solid-phase synthesis of variouspeptides are contained in separate solvent-permeable packets, enablingthe optimal use of the many identical repetitive steps involved insolid-phase methods. A completely manual procedure allows 500-1000 ormore syntheses to be conducted simultaneously. Houghten et al., supra,at 5134.

Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding epitope-bearing portions of the Ckβ-6protein.

In particular, such nucleic acid fragments of the Ckβ-6 of the presentinvention include nucleic acid molecules encoding: a polypeptidecomprising amino acid residues from about Val-12 to about Val-21 in FIG.1 (SEQ ID NO:2); a polypeptide comprising amino acid residues from aboutLeu-26 to about Lys-34 in FIG. 1 (SEQ ID NO:2); a polypeptide comprisingamino acid residues from about Phe-39 to about Cys-48 in FIG. 1 (SEQ IDNO:2); and a polypeptide comprising amino acid residues from aboutAsp-50 to about Gln-57 in FIG. 1 (SEQ ID NO:2); or any range or value inany of the foregoing.

Methods for determining other such epitope-bearing portions of an Ckβ-6polypeptide are described herein.

Epitope-bearing peptides and polypeptides of the invention are used toinduce antibodies according to methods well known in the art. See, forinstance, Sutcliffe, et al., supra; Wilson, et al., supra; Chow, M., etal., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. et al., J.Gen. Virol. 66:2347-2354 (1985). Generally, animals may be immunizedwith free peptide; however, anti-peptide antibody titer may be boostedby coupling of the peptide to a macromolecular carrier, such as keyholelimpet hemacyanin (KLH) or tetanus toxoid. For instance, peptidescontaining cysteine may be coupled to carrier using a linker such asm-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while otherpeptides may be coupled to carrier using a more general linking agentsuch as glutaraldehyde. Animals such as rabbits, rats and mice areimmunized with either free or carrier-cupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 g peptide or carrier protein and Freund's adjuvant. Severalbooster injections may be needed, for instance, at intervals of abouttwo weeks, to provide a useful titer of anti-peptide antibody which canbe detected, for example, by ELISA assay using free peptide adsorbed toa solid surface. The titer of anti-peptide antibodies in serum from animmunized animal may be increased by selection of anti-peptideantibodies, for instance, by adsorption to the peptide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

Immunogenic epitope-bearing peptides of the invention, i.e., those partsof a protein that elicit an antibody response when the whole protein isthe immunogen, are identified according to methods known in the art. Forinstance, Geysen et al., supra, discloses a procedure for rapidconcurrent synthesis on solid supports of hundreds of peptides ofsufficient purity to react in an enzyme-linked immunosorbent assay.Interaction of synthesized peptides with antibodies is then easilydetected without removing them from the support. In this manner apeptide bearing an immunogenic epitope of a desired protein may beidentified routinely by one of ordinary skill in the art. For instance,the immunologically important epitope in the coat protein offoot-and-mouth disease virus was located by Geysen et al. with aresolution of seven amino acids by synthesis of an overlapping set ofall 208 possible hexapeptides covering the entire 213 amino acidsequence of the protein. Then, a complete replacement set of peptides inwhich all 20 amino acids were substituted in turn at every positionwithin the epitope were synthesized, and the particular amino acidsconferring specificity for the reaction with antibody were determined.Thus, peptide analogs of the epitope-bearing peptides of the inventioncan be made routinely by this method. U.S. Pat. No. 4,708,781 to Geysen(1987) further describes this method of identifying a peptide bearing animmunogenic epitope of a desired protein.

Further still, U.S. Pat. No. 5,194,392 to Geysen (1990) describes ageneral method of detecting or determining the sequence of monomers(amino acids or other compounds) which is a topological equivalent ofthe epitope (i.e., a “mimotope”) which is complementary to a particularparatope (antigen binding site) of an antibody of interest. Moregenerally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a methodof detecting or determining a sequence of monomers which is atopographical equivalent of a ligand which is complementary to theligand binding site of a particular receptor of interest. Similarly,U.S. Pat. No. 5,480,971 to Houghten, R. A., et al. (1996) onPeralkylated Oligopeptide Mixtures discloses linear C1-C7-alkylperalkylated oligopeptides and sets and libraries of such peptides, aswell as methods for using such oligopeptide sets and libraries fordetermining the sequence of a peralkylated oligopeptide thatpreferentially binds to an acceptor molecule of interest. Thus,non-peptide analogs of the epitope-bearing peptides of the inventionalso can be made routinely by these methods.

The entire disclosure of each document cited in this section on“Polypeptides and Peptides” is hereby incorporated herein by reference.

As one of skill in the art will appreciate, Ckβ-6 polypeptides of thepresent invention and the epitope-bearing fragments thereof describedabove can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. This has been shown, e.g. for chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins (EPA 394,827; Traunecker, et al., Nature 331:84-86(1988)). Fusion proteins that have a disulfide-linked dimeric structuredue to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric Ckβ-6 protein or proteinfragment alone (Fountoulakis, et al., J. Biochem. 270:3958-3964 (1995)).

Vectors and Host Cells

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the genes of the present invention. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli lac or tryp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila S2 andSpodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma;adenoviruses; plant cells, etc. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

In addition to the use of expression vectors in the practice of thepresent invention, the present invention further includes novelexpression vectors comprising operator and promoter elements operativelylinked to nucleotide sequences encoding a protein of interest. Oneexample of such a vector is pHE4-5 (SEQ ID NO:21) which is described indetail below.

As summarized in FIGS. 21 and 22, components of the pHE4-5 vector (SEQID NOs:21 and 22) include: 1). a neomycinphosphotransferase gene as aselection marker, 2). an E. coli origin of replication, 3). a T5 phagepromoter sequence, 4). two lac operator sequences, 5). a nucleotidesequence encoding a Ckβ-6 polypeptide (for example, SEQ ID NOs:2 or 6),agonist or antagonist thereof, 6). a Shine-Delgarno sequence, 7). thelactose operon repressor gene (lacIq). The origin of replication (oriC)is derived from pUC19 (LTI, Gaithersburg, Md.). The promoter sequencewas and operator sequences were made synthetically. Synthetic productionof nucleic acid sequences is well known in the art. CLONTECH 95/96Catalog, pages 215-216, CLONTECH, 1020 East Meadow Circle, Palo Alto,Calif. 94303.

As noted above, the pHE4-5 vector contains a lacIq gene. LacIq is anallele of the lacI gene which confers tight regulation of the lacoperator. Amann, E. et al., Gene 69:301-315 (1988); Stark, M., Gene51:255-267 (1987). The lacIq gene encodes a repressor protein whichbinds to lac operator sequences and blocks transcription of down-stream(i.e., 3′) sequences. However, the lacIq gene product dissociates fromthe lac operator in the presence of either lactose or certain lactoseanalogs, e.g., isopropyl B-D-thiogalactopyranoside (IPTG). Thepolypeptide of the present invention thus is not produced in appreciablequantities in uninduced host cells containing the pHE4-5 vector.Induction of these host cells by the additional of an agent such asIPTG, however, results in the expression of the coding sequence for aCkβ-6, agonist or antagonists thereof.

The promoter/operator sequences of the pHE4-5 vector (SEQ ID NO:22)comprise a T5 phage promoter and two lac operator sequences. Oneoperator is located 5′ to the transcriptional start site and the otheris located 3′ to the same site. These operators, when present incombination with the lacIq gene product, confer tight repression ofdown-stream sequences in the absence of a lac operon inducer, e.g.,IPTG. Expression of operatively linked sequences located down-streamfrom the lac operators may be induced by the addition of a lac operoninducer, such as IPTG. Binding of a lac inducer to the lacIq proteinsresults in their release from the lac operator sequences and theinitiation of transcription of operatively linked sequences. Lac operonregulation of gene expression is reviewed in Devlin, T., TEXTBOOK OFBIOCHEMISTRY WITH CLINICAL CORRELATIONS, 4th Edition (1997), pages802-807.

The pHE4 series of vectors contain all of the components of the pHE4-5vector except for a coding sequence for a Ckβ-6, agonist or antagoniststhereof. Features of the pHE4 vectors include optimized synthetic T5phage promoter, lac operator, and Shine-Delagarno sequences. Further,these sequences are also optimally spaced so that expression of aninserted gene may be tightly regulated and high level of expressionoccurs upon induction.

Among known bacterial promoters suitable for use in the production ofproteins of the present invention include the E. coli lacI and lacZpromoters, the T3 and 17 promoters, the gpt promoter, the lambda PR andPL promoters and the trp promoter. Suitable eukaryotic promoters includethe CMV immediate early promoter, the HSV thymidine kinase promoter, theearly and late SV40 promoters, the promoters of retroviral LTRs, such asthose of the Rous Sarcoma Virus (RSV), and metallothionein promoters,such as the mouse metallothionein-I promoter.

The pHE4-5 vector also contains a Shine-Delgarno sequence 5′ to the AUGinitiation codon. Shine-Delgarno sequences are short sequences generallylocated about 10 nucleotides up-stream (i.e., 5′) from the AUGinitiation codon. These sequences essentially direct prokaryoticribosomes to the AUG initiation codon.

Thus, the present invention is also directed to expression vector usefulfor the production of the proteins of the present invention. This aspectof the invention is exemplified by the pHE4-5 vector (SEQ ID NO:21).

In addition, the present invention also includes recombinant constructscomprising one or more of the sequences as broadly described above. Theconstructs comprise a vector, such as a plasmid or viral vector, intowhich a sequence of the invention has been inserted, in a forward orreverse orientation. In a preferred aspect of this embodiment, theconstruct further comprises regulatory sequences, including, forexample, a promoter, operably linked to the sequence. Large numbers ofsuitable vectors and promoters are known to those of skill in the art,and are commercially available. The following vectors are provided byway of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10,phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A,pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis, L., et al., Basic Methods inMolecular Biology (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989), thedisclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Phar-macia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell known to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell23:175 (1981), and other cell lines capable of expressing a compatiblevector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.Mammalian expression vectors will comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation site, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking nontranscribedsequences. DNA sequences derived from the SV40 splice, andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

Polypeptide Purification and Isolation

The polypeptide can be recovered and purified from recombinant cellcultures by methods including ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps. A particularly preferred method of purification ofCkβ-6 polypeptides expressed in E. coli is described in Example 1,infra.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

Therapeutics

The polypeptide of the present invention can be used in a variety ofimmunoregulatory and inflammatory functions and also in a number ofdisease conditions. Ckβ-6 is in the chemokine family and therefore is achemo-attractant for leukocytes such as eosinophils, and basophils.

Northern blot analyses has shown that Ckβ-6 is expressed predominantlyin tissues of hematopoietic origin.

The polypeptide of the present invention, may be employed for thepromotion of wound healing. Since Ckβ-6 is a chemokine, it is achemo-attractant for leukocytes such as, basophils and eosinophilscausing infiltration of target immune cells to a wound area. In asimilar fashion, the polypeptides of the present invention can enhancehost defenses against chronic infections, e.g., mycobacterial, via theattraction and activation of microbicidal leukocytes.

The Ckβ-6 polypeptide may also be employed as an anti-tumor treatmentand for treating localized complications of a malignancy, such aspleural effusions or ascites. There is evidence that chemokineexpressing cells injected into tumors have caused regression of thetumor, for example, in the treatment of Karposi's sarcoma. Ckβ-6 mayinduce cells to secret TNF-α, which is a known agent for tumorregression. Ckβ-6 may also induce monocytes to secrete other tumor andcancer inhibiting agents-such as IL-6, IL-1 and G-CSF.

The presence of MCPs in vivo is accompanied by a local increase in thepresence of eosinophils which have the distinctive function of killingthe larvae of parasites that invade tissues, as in schistosomiasis,trichinosis and ascariasis. Therefore, Ckβ-6 may be employed forcombatting parasitic infections.

The polypeptide of the present invention may be employed for mobilizinghematopoietic progenitor cells into the peripheral blood circulation ofa non-human and human host, preferably a human host, for subsequentrecovery and use thereof in transplantation. The polypeptide of thepresent invention is administered in an amount effective to mobilizeinto and increase the amount of hematopoietic progenitor cells in theperipheral blood, in particular, increase the amount of humanhematopoietic stem cells in the peripheral blood. Such cells are oftenreferred to as CD34+ cells. For example, the polypeptide is administeredin amounts as hereinafter described. The polypeptide of the presentinvention may be administered alone or in conjunction with other agents,for example, GM-CSF and G-CSF which are known to be effective forincreasing such cells in peripheral blood. Mobilization of hematopoieticprogenitor cells into the peripheral circulation is important forautologous and heterologous bone marrow transfers which are used, forexample for treatment of cancer and hematological disorders.

The polypeptide of the present invention may also be employed to inhibitdestruction of hematopoietic progenitor cells in a non-human and humanhost, preferably a human host, resulting from treatment withchemotherapeutic agents. The polypeptide of the present invention may beadministered prior to, during or subsequent to chemotherapy and allows ahigher dose of chemotherapy to be employed in the treatment of cancer.The polypeptide of the present invention is administered in an amounteffective to inhibit destruction of hematopoietic progenitor cells; forexample, the polypeptide is administered in amounts as hereinafterdescribed. The polypeptide may be administered alone or in conjunctionwith other agents.

The hematopoietic cell protective compositions of the present inventionmay be used in combination with a variety of chemotherapeutic agentsincluding alkylating agents such as nitrogen mustards, ethylenimines,methylmelamines, alkyl sulfonates, nitrosuoureas, and triazenes;antimetabolites such as folic acid analogs, pyrimindine analogs, inparticular fluorocil and cytosine arabinoside, and purine analogs;natural products such as vinca alkaloids, epipodopyllotoxins,antibiotics, enzymes and biological response modifiers; andmiscellaneous products such as platinum coordination complexes,antracenedione, substituted urea such as hydroxyurea, methyl hydrazinederivatives, and adrenocorticoid suppresant.

Chemotherapeutic agents can be administered at known concentrationsaccording to known techniques. The protective compositions of thepresent invention can be co-administered with a chemotherapeutic agent,or administered separately, either before or after chemotherapeuticadministration.

The polypeptide of the present invention may also be employed to protecthematopoietic progenitor cells to thereby prevent or inhibit diseaseswhich may result from the destruction thereof; for example, leukopenia,myelo-dysplastic syndrome, and neutropenia.

The polypeptide of the present invention may also be employed in amountseffective to inhibit the degeneration of neuronal cells in non-human andhuman hosts, preferably a human host, which results from neuronaldegenerative diseases such as Alzheimer's disease, Parkinson's diseaseand AIDS-related complex. Neurodegenerative diseases include, but arenot limited to, AIDS dementia complex, demyelinating diseases, such asmultiple sclerosis and acute trasverse myelitis; extrapyramidal andcerebellar disorders such as lesions of the corticospinal system;disorders of the basal ganglia or cerebellar disorders; hyperkineticmovement disorders such as Huntington's Chorea and senile chorea;drug-induced movement disorders, such as those induced by drugs whichblock CNS dopamine receptors; hypokinetic movement disorders, such asParkinson's disease; Progressive supranucleo Palsy; structural lesionsof the cerebellum; spinocerebellar degenerations, such as spinal ataxia,Friedreich's ataxia, cerebellar cortical degenerations, multiple systemsdegenerations (Mencel, Dejerine-Thomas, Shi-Drager, and Machado-Joseph);systemic disorders (Refsum's disease, abetalipoprotemia, ataxia,telangiectasia, and mitochondrial multi.system disorder); demyelinatingcore disorders, such as multiple sclerosis, acute transverse myelitis;and disorders of the motor unit such as neurogenic muscular atrophies(anterior horn cell degeneration, such as amyotrophic lateral sclerosis,infantile spinal muscular atrophy and juvenile spinal muscular atrophy);Alzheimer's disease; Down's Syndrome in middle age; Diffuse Lewy bodydisease; Senile Dementia of Lewy body type; Wernicke-Korsakoff syndrome;chronic alcoholism; Creutzfeldt-Jakob disease; Subacute sclerosingpanencephalitis Hallerrorden-Spatz disease; and Dementia pugilistica.One preferred neurodegenerative disease is multiple sclerosis. Forexample, the polypeptide may be employed in amounts as hereinafterdescribed.

In addition, recent demonstration that the MIP-1 a receptor serves as acofactor in facilitating the entry of HIV into human monocytes andT-lymphocytes raises an interesting possibility that Ckβ-6 or itsvariants might interfere with the process of HIV entry, or the entry ofother viruses, particularly retroviruses, into cells. The, Ckβ-6 can beuseful as an antiviral agent for viruses and retroviruses whose entry isfacilitated by the Ckβ-6 receptor.

TABLE 2 Effect of Ckβ-6 administration to mice on the distribution ofthe primitive hematopoietic progenitors in peripheral blood, spleen, andbone marrow after two days Numbers of Progenitors per 10⁴ PB cells 10⁴Spleen cells 10⁴ BM cells Treatment HPP LPP IM HPP LPP IM HPP LPP Saline0.5 38 6.5 0.7 5.5 1.5 53 484 ±0.7 ±9.5 ±1.9 ±1.5 ±2.5 ±2.3 ±11 ±59Ckβ-6 3.5 95 25 2.75 4.2 3.5 27 610 (1 mg/kg/ ±0.5 ±16.9 ±13.5 ±0.9 ±3.5±2.4 ±3.5 ±28 day) PB = Peripheral blood, mononuclear cells Spl. = Lowdensity fraction of spleen cells BM = Bone marrow fraction that is6-fold enriched for the primitive cells HPP = High proliferativepotential colony forming cells LPP = Low proliferative potential colonyforming cells IM = Immature cell, a rare cell type found in the bonemarrow, gives rise to a highly refrectile, small (<50 cells/colony)colony in the presence of multiple cytokines; the cells within thecolony are stacked in a horizontal plane and they exhibit blast celllike nuclear staining characteristics.

Three mice were injected IP daily with either Ckβ-6 or saline. Fortyeight hours after the first injection, blood was collected from eachanimal by cardiac puncture and mice were then sacrificed to obtain bonemarrow and spleen. Indicated numbers of cells from each of the tissueswere then plated in duplicates in agar-containing medium in the presenceof rmIL-3(5 ng/ml), rmSCF(50 ng/ml), rhM-CSF(5 ng/ml), and rmIL-1a(10ng/ml) and incubated for 14 days. Data are pooled from three animals ineach group and expressed as mean±S.D.

TABLE 3 Effect of Ckβ-6 administration to mice on the distribution ofthe primitive hematopoietic progenitors in peripheral blood, spleen, andbone marrow after four days Numbers of Progenitors per 10⁴ PB cells 10⁴Spleen cells 10⁴ BM cells Treatment HPP LPP IM HPP LPP IM HPP LPP Saline0 29 1 1 10 0.8 60 505 ±5.6 ±1.5 ±0.6 ±4.6 ±0.7 ±8 ±45 Ckβ- 6 3.8 84.528.6 2.6 10.3 7 26.5 330 (1 mg/kg/ ±1.5 ±14.5 ±8.6 ±0.5 ±2.1 ±1.5 ±8 ±46day) PB = Peripheral blood, mononuclear cells Spl. = Low densityfraction of spleen cells BM = Bone marrow fraction that is 6-foldenriched for the primitive cells HPP = High proliferative potentialcolony forming cells LPP = Low proliferative potential colony formingcells IM = Immature cell, a rare cell type found in the bone marrow,gives rise to a highly refrectile, small (<50 cells/colony) colony inthe presence of multiple cytokines; the cells within the colony arestacked in a horizontal plane and they exhibit blast cell like nuclearstaining characteristics.

Three mice were injected IP daily with either Ckβ-6 or saline. Ninetysix hours after the first injection, blood was collected from eachanimal by cardiac puncture and mice were then sacrificed to obtain bonemarrow and spleen. Indicated numbers of cells from each of the tissueswere then plated in duplicates in agar-containing medium in the presenceof rmIL-3(5 ng/ml), rmSCF(50 ng/ml), rhM-CSF(5 ng/ml), and rmIL-1a(10ng/ml) and incubated for 14 days. Data are pooled from three animals ineach group and expressed as mean±S.D.

TABLE 4 Analysis of the peripheral blood leukocyte composition by FACSanin mice administered with Ckβ-6 after two days Percent Positive in theGated the Cell Populations CD45R + GR.1 + Mac. 1 + CD8 + CD4 + TreatmentB-Cells PMN Monocytes T-cells T-cells Saline 40.5 ± 9.2 62.5 ± 10.6 19.5± 2.1 29 ± 5.6 39 ± 12 Ckβ-6   37 ± 5.6   56 ± 11.3   18 ± 4.2 27 ± 4.333 ± 7  (mg/Kg/ day)

Three C57 Black 6 mice (˜20 g weight) were injected (IP) daily witheither saline or Ckβ-6. Forty eight hours after the first injection,blood was collected by cardiac puncture and mice were sacrificed toobtain spleen and bone marrow cells. For immunostaining, 0.1 ml of bloodfrom each of the animal was first treated with Gen Trak lysing solutionto lyse the red blood cells. Nucleated cells were then sedimented,washed with PBS, and incubated with PE-conjugated monoclonal antibodiesagainst CD45R, Gr. 1, Mac. 1, CD4, & CD8 and processed forflowcytometry. At least 10,000 cells were analyzed. Data are expressedas mean percent positive cells in the appropriate channels±SD.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents for in vitro purposes related toscientific research, synthesis of DNA and manufacture of DNA vectors,and for the purpose of developing therapeutics and for the treatment ofhuman disease. For example, Ckβ-6 may be employed for the expansion ofimmature hematopoietic progenitor cells, for example, granulocytes,macrophages or monocytes, by temporarily preventing theirdifferentiation. These bone marrow cells may be cultured in vitro.

Receptors

This invention provides a method for identification of the receptor forCkβ-6. The gene encoding the receptor can be identified by numerousmethods known to those of skill in the art, for example, ligand panningand FACS sorting (Coligan, et al., Current Protocols in Immun., 1(2),Chapter 5, (1991)). Preferably, expression cloning is employed whereinpolyadenylated RNA is prepared from a cell responsive to Ckβ-6, and acDNA library created from this RNA is divided into pools and used totransfect COS cells or other cells that are not responsive to Ckβ-6.Transfected cells which are grown on glass slides are exposed to labeledCkβ-6. Ckβ-6 can be labeled by a variety of means including iodinationor inclusion of a recognition site for a site-specific protein kinase.Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clone that encodesthe putative receptor. As an alternative approach for receptoridentification, labeled ligand can be photoaffinity linked with cellmembrane or extract preparations that express the receptor molecule.Cross-linked material is resolved by PAGE and exposed to X-ray film. Thelabeled complex containing the ligand-receptor can be excised, resolvedinto peptide fragments, and subjected to protein microsequencing. Theamino acid sequence obtained from microsequencing would be used todesign a set of degenerate oligonucleotide probes to screen a cDNAlibrary to identify the gene encoding the putative receptor.

Chemokine Receptor-3 (CCR3) has been identified as one of the Ckβ-6receptors herein (see Examples 9, 10 and 11, below). CCR3 is also knownto be a receptor for chemokine-β-10 (published as “MCP-4” in Uguccioni,M., et al., J. Exp. Med. 183:2379-2384 (1996)) and eotaxin. Accordingly,as would be expected by those of skill in the art, Ckβ-6 antagonistswhich are capable of binding to CCR3 but lack the capacity to inducesignal transduction would also be expected to be antagonists ofchemokine-β-10 activity and eotaxin activity. Such activities aredescribed below.

Antagonists, Agonists and Methods

This invention also provides a method of screening compounds to identifyagonists and antagonists to the polypeptide of the present invention. Anagonist is a compound which has similar biological functions, orenhances the functions, of the polypeptides, while antagonists blocksuch functions. As an example, a mammalian cell or membrane preparationexpressing an Ckβ-6 receptor would be contacted with a compound ofinterest. The ability of the compound to generate a the response of aknown second messenger system following interaction with the Ckβ-6receptor is then measured. Such second messenger systems include but arenot limited to, calcium release (as described, for example, in Example9, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.The ability of a compound to bind the Ckβ-6 receptor and elicit a secondmessenger response identifies that compound as an agonist. A compoundwhich binds but does not elicit a second messenger response identifiesthat compound as an antagonist.

A competitive binding assay, in which the compounds are labeled, forexample by radioactivity may also be employed to identify antagonists.Such methods are known in the art.

Antagonists include negative dominant mutants of Ckβ-6. Ckβ-6 is atetrameric polypeptide wherein one mutated unit will cause the entirepolypeptide to be non-functional. A negative dominant mutant of Ckβ-6binds to the Ckβ-6 receptor but fails to activate cells (leukocytes andeosinophils) to which it binds. An assay to detect negative dominantmutants of Ckβ-6 is an in vitro chemotaxis assay wherein a multiwellchemotaxis chamber equipped with polyvinylpyrrolidone-free polycarbonatemembranes is used to measure the chemoattractant ability of Ckβ-6 forleukocytes in the presence and absence of potential antagonist oragonist molecules, such as is described in Example 10, below. Apreferred assay is an in-vitro calcium (Ca²⁺) release assay, forexample, as described in Example 9, below.

Potential antagonists also include an antibody, or in some cases, anoligopeptide or oligonucleotide, which binds to the polypeptide andprevents it from binding its receptor.

Another potential antagonist is an antisense construct prepared usingantisense technology. Antisense technology can be used to control geneexpression through triple-helix formation or antisense DNA or RNA, bothof which methods are based on binding of a polynucleotide to DNA or RNA.For example, the 5′ coding portion of the polynucleotide sequence, whichencodes for the mature polypeptides of the present invention, is used todesign an antisense RNA oligonucleotide of from about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription (triple helix—see Lee,et al., Nucl. Acids Res. 6:3073 (1979); Cooney, et al., Science 241:456(1988); and Dervan, et al., Science 251:1360 (1991)), thereby preventingtranscription and the production of Ckβ-6. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into Ckβ-6 polypeptide (Antisense—Okano, J. Neurochem.56:560 (1991); Oligodeoxnucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988)). The oligonucleotidesdescribed above can also be delivered to cells such that the antisenseRNA or DNA may be expressed in vivo to inhibit production of Ckβ-6.

Potential antagonists include a small molecule which binds to andoccupies the active site of the polypeptide thereby making the catalyticsite inaccessible to substrate such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall peptides or peptide-like molecules.

Another potential antagonist is a peptide derivative of the polypeptideswhich are naturally or synthetically modified analogs of thepolypeptides that have lost biological function yet still recognize andbind to the receptors of the polypeptides to thereby effectively blockthe receptors. Examples of peptide derivatives include, but- are notlimited to small peptides or peptide-like molecules.

The antagonists may be employed to treat disorders which are eitherCkβ-6-induced or enhanced, for example, autoimmune and chronicinflammatory and infective diseases. Examples of auto-immune diseasesinclude multiple sclerosis, and insulin-dependent diabetes.

The antagonists may be employed to treat inflammation by preventing theattraction of eosinophils or basophiles to a wound or a site of trauma,and to regulate normal pulmonary macrophage populations, since acute andchronic inflammatory pulmonary diseases are associated withsequestration of mononuclear phagocytes in the lung. They may also beemployed to treat rheumatoid arthritis, since MCP levels were found tobe significantly elevated in synovial fluid from rheumatoid arthritispatients which suggests that synovial production of Ckβ-6 attractseosinophils or basophils whose influx and activation are important inthe pathogenesis of both degenerative and inflammatory arthropathies.

The antagonists may also be employed to prevent allergies, since it hasbeen shown that MCPs directly induce histamine release by basophils.Related immunological disorders including late phase allergic reactions,chronic urticaria, and atopic dermatitis can be treated by antagonistswhich are effective to inhibit chemokine-induced mast cell and basophildegranulation and release of histamine. IgE-mediated allergic reactionssuch as asthma, rhinitis, and eczema may also be treated. Antagonist canalso be used to treat adult respiratory distress syndrome as well asairway inflammation.

Antagonists may also be employed to treat idiopathic hyper-eosinophilicsyndrome by preventing eosinophil production an migration. Endotoxicshock may also be treated by the antagonists by preventing the migrationof macrophages and their production of the chemokine polypeptides of thepresent invention. The antagonists may be employed in a composition witha pharmaceutically acceptable carrier, e.g., as herein described.

Antagonists may also be employed to treat rheumatoid arthritis bypreventing the attraction of eosinophils and basophils into synovialfluid in the joints of patients.

The antagonists may be employed to interfere with the deleteriouscascades attributed primarily to IL-1 and TNF, which prevents thebiosynthesis of other inflammatory cytokines. In this way, theantagonists may be employed to inhibit prostaglandin-independent feverinduced by chemokines.

The antagonists may also be employed to treat bone marrow failure, forexample, aplastic anemia and myelodysplastic syndrome. The antagonistsmay also be employed to treat subepithelial basement membrane fibrosiswhich is a prominent feature of the asthmatic lung.

Ckβ-6 agonists include any small molecule that has an activity similarto Ckβ-6 polypeptides, as described herein. For example, Ckβ-6 agonistscan be used to enhance Ckβ-6 activity. For example, to enhance Ckβ-6induced myeloprotection in patients undergoing chemotherapy or bonemarrow transplantation.

Pharmaceutical Compositions

The Ckβ-6 pharmaceutical composition comprises an effective amount of anisolated Ckβ-6 polypeptide, agonist or antagonist of the invention,particularly a mature form of Ckβ-6, effective to increase the Ckβ-6activity level in such an individual. Such compositions can beformulated and doses in a fashion consistent with good medical practice,taking into account the clinical condition of the individual patient(especially the side effects of treatment with Ckβ-6 polypeptide alone),the site of delivery of the polypeptide composition, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The effective amount of Ckβ-6 polypeptide forpurposes herein is thus determined by such considerations.

The polypeptides, and agonists and antagonists, of the present inventionmay be employed in combination with a suitable pharmaceutical carrier.Such compositions comprise a therapeutically effective amount of thepolypeptide or agonist or antagonist, and a pharmaceutically acceptablecarrier or excipient. Such a carrier includes but is not limited tosaline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. The formulation should suit the mode ofadministration.

By “pharmaceutically acceptable carrier” is meant a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The term “parenteral” as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

The Ckβ-6 polypeptide is also suitably administered by sustained-releasesystems. Suitable examples of sustained-release compositions includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or mirocapsules. Sustained-release matrices include polylactides(U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman, U., et al., Biopolymers 22:547-556(1983)), poly (2-hydroxyethyl methacylate) (R. Langer, et al., J.Biomed. Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech.12:98-105 (1982)), ethylene vinyl acetate (R. Langer, et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustained-release Ckβ-6polypeptide compositions also include liposomally entrapped Ckβ-6polypeptide. Liposomes containing Ckβ-6 polypeptide are prepared bymethods known per se: DE 3,218,121; Epstein, et al., Proc. Natl. Acad.Sci. (USA) 82:3688-3692 (1985); Hwang, et al., Proc. Natl. Acad. Sci.(USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949;EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. percent cholesterol, the selected proportionbeing adjusted for the optimal Ckβ-6 polypeptide therapy.

For parenteral administration, in one embodiment, the Ckβ-6 polypeptideis formulated generally by mixing it at the desired degree of purity, ina unit dosage injectable form (solution, suspension, or emulsion), witha pharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to polypeptides.

Generally, the formulations are prepared by contacting the Ckβ-6polypeptide uniformly and intimately with liquid carriers or finelydivided solid carriers or both. Then, if necessary, the product isshaped into the desired formulation. Preferably the carrier is aparenteral carrier, more preferably a solution that is isotonic with theblood of the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, and dextrose solution. Non-aqueous vehiclessuch as fixed oils and ethyl oleate are also useful herein, as well asliposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g. polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

The Ckβ-6 polypeptide is typically formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, ata pH of about 3 to 8. It will be-understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of Ckβ-6 polypeptide salts.

Ckβ-6 polypeptide to be used for therapeutic administration must besterile. Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g. 0.2 micron membranes). Therapeutic Ckβ-6polypeptide compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

Ckβ-6 polypeptide ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. As anexample of a lyophilized formulation, 10-ml vials are filled with 5 mlof sterile-filtered 1% (w/v) aqueous Ckβ-6 polypeptide solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized Ckβ-6 polypeptide using bacteriostaticWater-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides, or agonists and antagonists, of the present invention maybe employed in conjunction with other therapeutic compounds.

Modes of Administration

It will be appreciated that conditions caused by a decrease in thestandard or normal level of Ckβ-6 activity in an individual, can betreated by administration of Ckβ-6 protein. Thus, the invention furtherprovides a method of treating an individual in need of an increasedlevel of Ckβ-6 activity comprising administering to such an individual apharmaceutical composition comprising an effective amount of an isolatedCkβ-6 polypeptide of the invention, particularly a mature form of theCkβ-6, effective to increase the Ckβ-6 activity level in such anindividual.

The amounts and dosage regimens of Ckβ-6 administered to a subject willdepend on a number of factors such as the mode of administration, thenature of the condition being treated and the judgment of theprescribing physician. The pharmaceutical compositions are administeredin an amount which is effective for treating and/or prophylaxis of thespecific indication. In general, the polypeptides will be administeredin an amount of at least about 10 μg/kg body weight and in most casesthey will be administered in an amount not in excess of about 10 mg/kgbody weight per day and preferably the dosage is from about 10 μg/kgbody weight daily, taking into account the routes of administration,symptoms, etc.

As a general proposition, the total pharmaceutically effective amount ofCkβ-6 polypeptide administered parenterally per dose will morepreferably be in the range of about 1 μg/kg/day to 10 mg/kg/day ofpatient body weight, although, as noted above, this will be subject totherapeutic discretion. Even more preferably, this dose is at least 0.01mg/kg/day, and most preferably for humans between about 0.01 and 1mg/kg/day. If given continuously, the Ckβ-6 polypeptide is typicallyadministered at a dose rate of about 1 μg/kg/hour to about 50 μg/kghour, either by 1-4 injections per day or by continuous subcutaneousinfusions, for example, using a mini-pump. An intravenous bag solutionmay also be employed. The length of treatment needed to observe changesand the interval following treatment for responses to occur appears tovary depending on the desired effect.

The pharmaceutical compositions may be administered in a convenientmanner such as by the oral, topical, parenterally, intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal or intradermalroutes.

Gene Therapy

The polypeptides and agonists and antagonists which are polypeptides mayalso be employed in accordance with the present invention by expressionof such polypeptides in vivo, which is often referred to as “genetherapy.”

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art and are apparentfrom the teachings herein. For example, cells may be engineered by theuse of a retroviral plasmid vector containing RNA encoding a polypeptideof the present invention.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Forexample, a packaging cell is transduced with a retroviral plasmid vectorcontaining RNA encoding a polypeptide of the present invention such thatthe packaging cell now produces infectious viral particles containingthe gene of interest. These producer cells may be administered to apatient for engineering cells in vivo and expression of the polypeptidein vivo. These and other methods for administering a polypeptide of thepresent invention by such method should be apparent to those skilled inthe art from the teachings of the present invention.

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

In a preferred embodiment the retroviral expression vector, pMV-7, isflanked by the long terminal repeats (LTRs) of the Moloney murinesarcoma virus and contains the selectable drug resistance gene neo underthe regulation of the herpes simplex virus (HSV) thymidine kinase (tk)promoter. Unique EcoRI and HindIII sites facilitate the introduction ofcoding sequence (Kirschmeier, P. T., et al., DNA 7:219-25 (1988)).

The vector includes one or more promoters. Suitable promoters which maybe employed include, but are not limited to, the retrovial LTR; the SV40promoter; and the human cytomegalovirus (CMV) promoter described inMiller, et al., Biotechniques 7(9):980-990 (1989), or any other promoter(e.g., cellular promoters such as eukaryotic cellular promotersincluding, but not limited to, the histone, pol III, and b-actinpromoters). Other viral promoters which may be employed include, but arenot limited to, adenovirus promoters, thymidine kinase (TK) promoters,and B19 parvovirus promoters. The selection of a suitable promoter willbe apparent to those skilled in the art from the teachings containedherein.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the b-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, y-2,y-AM, PA12, T19-14X, VT-19-17-H2, yCRE, yCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy, Vol. 1(1990), pp. 5-14, which is incorporated herein by reference in itsentirety. The vector may transduce the packaging cells through any meansknown in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO₄ precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the polypeptides.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

Disease Diagnosis and Prognosis

Certain diseases or disorders, as discussed below, may be associatedwith altered (enhanced or reduced) levels of the Ckβ-6 protein and mRNAencoding the Ckβ-6 protein when compared to a corresponding “standard”mammal, i.e., a mammal of the same species not having the disease ordisorder. Further, it is believed that altered levels of the Ckβ-6protein can be detected in certain body fluids (e.g. sera, plasma,urine, and spinal fluid) from mammals with a disease or disorder whencompared to sera from mammals of the same species not having the diseaseor disorder. Thus, the invention provides a diagnostic method, whichinvolves assaying the expression level of the gene encoding the Ckβ-6protein in mammalian cells or body fluid and comparing the geneexpression level with a standard Ckβ-6 gene expression level, whereby analteration in the gene expression level compared to the standard isindicative of certain diseases or disorders.

Where a disease or disorder diagnosis has already been made according toconventional methods, the present invention is useful as a prognosticindicator, whereby patients exhibiting altered Ckβ-6 gene expressionwill experience a worse clinical outcome relative to patients expressingthe gene at a level closer to normal.

By “assaying the expression level of the gene encoding the Ck4-6protein” is intended qualitatively or quantitatively measuring orestimating the level of the Ckβ-6 protein or the level of the mRNAencoding the Ckβ-6 protein in a first biological sample either directly(e.g. by determining or estimating absolute protein level or mRNA level)or relatively (e.g. by comparing to the Ckβ-6 protein level or mRNAlevel in a second biological sample).

Preferably, the Ckβ-6 protein level or mRNA level in the firstbiological sample is measured or estimated and compared to a standardCkβ-6 protein level or mRNA level, the standard being taken from asecond biological sample obtained from an individual not having thedisease or disorder. As will be appreciated in the art, once a standardCkβ-6 protein level or mRNA level is known, it can be used repeatedly asa standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, cell line, tissue culture, or other source which containsCkβ-6 protein or mRNA. Biological samples include mammalian body fluids(such as sera, plasma, urine, synovial fluid and spinal fluid) whichcontain secreted mature Cβ-6 protein, and haematopoietic tissue. Methodsfor obtaining tissue biopsies and body fluids from mammals are wellknown in the art. Where the biological sample is to include mRNA, atissue biopsy is the preferred source.

The present invention is useful for detecting disease in mammals. Inparticular the invention is useful during useful for diagnosis ortreatment of various immune system-related disorders in mammals,preferably humans. Such disorders include tumors, cancers, and anydisregulation of immune cell function including, but not limited to,autoimmunity, arthritis, leukemias, lymphomas, immunosupression, sepsis,wound healing, acute and chronic infection, cell mediated immunity,humoral immunity, inflammatory bowel disease, myelosupression, asthmaand the like. Preferred mammals include monkeys, apes, cats, dogs, cows,pigs, horses, rabbits and humans. Particularly preferred are humans.

Total cellular RNA can be isolated from a biological sample using anysuitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described inChomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels ofmRNA encoding the Ckβ-6 protein are then assayed using any appropriatemethod. These include Northern blot analysis, S1 nuclease mapping, thepolymerase chain reaction (PCR), reverse transcription in combinationwith the-polymerase chain reaction (RT-PCR), and reverse transcriptionin combination with the ligase chain reaction (RT-LCR).

Northern blot analysis can be performed as described in Harada, et al.,Cell 63:303-312 (1990). Briefly, total RNA is prepared from a biologicalsample as described above. For the Northern blot, the RNA is denaturedin an appropriate buffer (such as glyoxal/dimethyl sulfoxide/sodiumphosphate buffer), subjected to agarose gel electrophoresis, andtransferred onto a nitrocellulose filter. After the RNAs have beenlinked to the filter by a UV linker, the filter is prehybridized in asolution containing formamide, SSC, Denhardt's solution, denaturedsalmon sperm, SDS, and sodium phosphate buffer. Ckβ-6 protein cDNAlabeled according to any appropriate method (such as the 32P-multiprimedDNA labeling system (Amersham)) is used as probe. After hybridizationovernight, the filter is washed and exposed to x-ray film. cDNA for useas probe according to the present invention is described in the sectionsabove and will preferably at least 15 bp in length.

S1 mapping can be performed as described in Fujita, et al., Cell49:357-367 (1987). To prepare probe DNA for use in S1 mapping, the sensestrand of above-described cDNA is used as a template to synthesizelabeled antisense DNA. The antisense DNA can then be digested using anappropriate restriction endonuclease to generate further DNA probes of adesired length. Such antisense probes are useful for visualizingprotected bands corresponding to the target mRNA (i.e., mRNA encodingthe Ckβ-6 protein). Northern blot analysis can be performed as describedabove.

Preferably, levels of mRNA encoding the Ckβ-6 protein are assayed usingthe RT-PCR method described in Makino, et al., Technique 2:295-301(1990). By this method, the radioactivities of the “amplicons” in thepolyacrylamide gel bands are linearly related to the initialconcentration of the target mRNA. Briefly, this method involves addingtotal RNA isolated from a biological sample in a reaction mixturecontaining a RT primer and appropriate buffer. After incubating forprimer annealing, the mixture can be supplemented with a RT buffer,dNTPs, DTT, RNase inhibitor and reverse transcriptase. After incubationto achieve reverse transcription of the RNA, the RT products are thensubject to PCR using labeled primers. Alternatively, rather thanlabeling the primers, a labeled dNTP can be included in the PCR reactionmixture. PCR amplification can be performed in a DNA thermal cycleraccording to conventional techniques. After a suitable number of roundsto achieve amplification, the PCR reaction mixture is electrophoresed ona polyacrylamide gel. After drying the gel, the radioactivity of theappropriate bands (corresponding to the mRNA encoding the Ckβ-6protein)) is quantified using an imaging analyzer. RT and PCR reactioningredients and conditions, reagent and gel concentrations, and labelingmethods are well known in the art. Variations on the RT-PCR method willbe apparent to the skilled artisan.

Any set of oligonucleotide primers which will amplify reversetranscribed target mRNA can be used and can be designed as described inthe sections above.

Assaying Ckβ-6 protein levels in a biological sample can occur using anyart-known method. Preferred for assaying Ckβ-6 protein levels in abiological sample are antibody-based techniques. For example, Ckβ-6protein expression in tissues can be studied with classicalimmunohistological methods. In these, the specific recognition isprovided by the primary antibody (polyclonal or monoclonal) but thesecondary detection system can utilize fluorescent, enzyme, or otherconjugated secondary antibodies. As a result, an immunohistologicalstaining of tissue section for pathological examination is obtained.Tissues can also be extracted, e.g. with urea and neutral detergent, forthe liberation of Ckβ-6 protein for Western-blot or dot/slot assay(Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M.,et al., J. Cell. Biol. 105:3087-3096 (1987)). In this technique, whichis based on the use of cationic solid phases, quantitation of Ckβ-6protein can be accomplished using isolated Ckβ-6 protein as a standard.This technique can also be applied to body fluids. With these samples, amolar concentration of Ckβ-6 protein will aid to set standard values ofCkβ-6 protein content for different body fluids, like serum, plasma,urine, spinal fluid, etc. The normal appearance of Ckβ-6 protein amountscan then be set using values from healthy individuals, which can becompared to those obtained from a test subject.

Other antibody-based methods useful for detecting Ckβ-6 protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). For example, an Ckβ-6protein-specific monoclonal antibodies can be used both as animmunoabsorbent and as an enzyme-labeled probe to detect and quantifythe Ckβ-6 protein. The amount of Ckβ-6 protein present in the sample canbe calculated by reference to the amount present in a standardpreparation using a linear regression computer algorithm. In anotherELISA assay, two distinct specific monoclonal antibodies can be used todetect Ckβ-6 protein in a body fluid. In this assay, one of theantibodies is used as the immunoabsorbent and the other as theenzyme-labeled probe.

The above techniques may be conducted essentially as a “one-step” or“two-step” assay. The “one-step” assay involves contacting Ckβ-6 proteinwith immobilized antibody and, without washing, contacting the mixturewith the labeled antibody. The “two-step” assay involves washing beforecontacting the mixture with the labeled antibody. Other conventionalmethods may also be employed as suitable. It is usually desirable toimmobilize one component of the assay system on a support, therebyallowing other components of the system to be brought into contact withthe component and readily removed from the sample.

Suitable enzyme labels include, for example, those from the oxidasegroup, which catalyze the production of hydrogen peroxide by reactingwith substrate. Glucose oxidase is particularly preferred as it has goodstability and its substrate (glucose) is readily available. Activity ofan oxidase label may be assayed by measuring the concentration ofhydrogen peroxide formed by the enzyme-labelled antibody/substratereaction. Besides enzymes, other suitable labels include radioisotopes,such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H),indium (¹¹²In), and technetium (⁹⁹mTc), and fluorescent labels, such asfluorescein and rhodamine, and biotin.

The polypeptides of the present invention, and polynucleotides encodingsuch polypeptides, may be employed as research reagents for in vitropurposes related to scientific research, synthesis of DNA andmanufacture of DNA vectors, and for the purpose of developingtherapeutics and diagnostics for the treatment of human disease. Forexample, Ckβ-6 may be employed for the expansion of immaturehematopoietic progenitor cells, for example, granulocytes, macrophagesor monocytes, by temporarily preventing their differentiation. Thesebone marrow cells may be cultured in vitro.

Fragments of the full length Ckβ-6 genes may be used as a hybridizationprobe for a cDNA library to isolate the full length gene and to isolateother genes which have a high sequence similarity to the gene or similarbiological activity. Preferably, however, the probes have at least 30bases and may contain, for example, 50 or more bases. The probe may alsobe used to identify a cDNA clone corresponding to a full lengthtranscript and a genomic clone or clones that contain the complete genesincluding regulatory and promotor regions, exons, and introns. Anexample of a screen comprises isolating the coding region of the genesby using the known DNA sequence to synthesize an oligonucleotide probe.Labeled oligonucleotides having a sequence complementary to that of thegenes of the present invention are used to screen a library of humancDNA, genomic DNA or mRNA to determine which members of the library theprobe hybridizes to.

This invention is also related to the use of the gene of the presentinvention as a diagnostic. Detection of a mutated form of the gene willallow a diagnosis of a disease or a susceptibility to a disease whichresults from underexpression of Ckβ-6.

Individuals carrying mutations in the gene of the present invention maybe detected at the DNA level by a variety of techniques. Nucleic acidsfor diagnosis may be obtained from a patient's cells, including but notlimited to blood, urine, saliva, tissue biopsy and autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR (Saiki et al., Nature 324:163-166 (1986))prior to analysis. RNA or cDNA may also be used for the same purpose. Asan example, PCR primers complementary to the nucleic acid encoding Ckβ-6can be used to identify and analyze mutations. For example, deletionsand insertions can be detected by a change in size of the amplifiedproduct in comparison to the normal genotype. Point mutations can beidentified by hybridizing amplified DNA to radiolabeled RNA oralternatively, radiolabeled antisense DNA sequences. Perfectly matchedsequences can be distinguished from mismatched duplexes by RNase Adigestion or by differences in melting temperatures.

Sequence differences between the reference gene and genes havingmutations may be revealed by the direct DNA sequencing method. Inaddition, cloned DNA segments may be employed as probes to detectspecific DNA segments. The sensitivity of this method is greatlyenhanced when combined with PCR. For example, a sequencing primer isused with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA flagments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science 230:1242 (1985)).

Sequence changes at specific locations. may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci. (USA)85:43974401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic assay for detectingaltered levels of the polypeptide of the present invention in varioustissues since an over-expression of the proteins compared to normalcontrol tissue samples may detect the presence of a disease orsusceptibility to a disease, for example, a tumor. Assays used to detectlevels of the polypeptide of the present invention in a sample derivedfrom a host are well-known to those of skill in the art and includeradioimmunoassays, competitive-binding assays, Western Blot analysis,ELISA assays and sandwich assays. An ELISA assay (Coligan, et al.,Current Protocols in Immunology 1(2), Chapter 6, (1991)) initiallycomprises preparing an antibody specific to the Ckβ-6 antigen,preferably a monoclonal antibody. In addition a reporter antibody isprepared against the monoclonal antibody. To the reporter antibody isattached a detectable reagent such as radioactivity, fluorescence or inthis example a horseradish peroxidase enzyme. A sample is now removedfrom a host and incubated on a solid support, e.g. a polystyrene dish,that binds the proteins in the sample. Any free protein binding sites onthe dish are then covered by incubating with a non-specific protein suchas bovine serum albumin. Next, the monoclonal antibody is incubated inthe dish during which time the monoclonal antibodies attached to any ofthe polypeptide of the present invention attached to the polystyrenedish. All unbound monoclonal antibody is washed out with buffer. Thereporter antibody linked to horseradish peroxidase is now placed in thedish resulting in binding of the reporter antibody to any monoclonalantibody bound to the polypeptide of the present invention. Unattachedreporter antibody is then washed out. Peroxidase substrates are thenadded to the dish and the amount of color developed in a given timeperiod is a measurement of the amount of the polypeptide of the presentinvention present in a given volume of patient sample when comparedagainst a standard curve.

A competition assay may be employed wherein antibodies specific to thepolypeptide of the present invention are attached to a solid support andlabeled Ckβ-6 and a sample derived from the host are passed over thesolid support and the amount of label detected attached to the solidsupport can be correlated to a quantity of the polypeptide of thepresent invention in the sample.

A “sandwich” assay is similar to an ELISA assay. In a “sandwich” assayCkβ-6 is passed over a solid support and binds to antibody attached to asolid support. A second antibody is then bound to the Ckβ-6. A thirdantibody which is labeled and specific to the second antibody is thenpassed over the solid support and binds to the second antibody and anamount can then be quantified.

This invention provides a method for identification of the receptors forthe chemokine polypeptides. The gene encoding the receptor can beidentified by numerous methods known to those of skill in the art, forexample, ligand panning and FACS sorting (Coligan, et al., CurrentProtocols in Immun. 1(2), Chapter 5, (1991)). Preferably, expressioncloning is employed wherein polyadenylated RNA is prepared from a cellresponsive to the polypeptides, and a cDNA library created from this RNAis divided into pools and used to transfect COS cells or other cellsthat are not responsive to the polypeptides. Transfected cells which aregrown on glass slides are exposed to the labeled polypeptides. Thepolypeptides can be labeled by a variety of means including iodinationor inclusion of a recognition site for a site-specific protein kinase.Following fixation and incubation, the slides are subjected toautoradiographic analysis. Positive pools are identified and sub-poolsare prepared and retransfected using an iterative sub-pooling andrescreening process, eventually yielding a single clones that encodesthe putative receptor.

As an alternative approach for receptor identification, the labeledpolypeptides can be photoaffinity linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE analysis and exposed to X-ray film. The labeledcomplex containing the receptors of the polypeptides can be excised,resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

Chromosome Assays

The nucleic acids of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of an Ckβ-6 protein gene. Thiscan be accomplished using a variety of well known techniques andlibraries, which are generally available commercially. The genomic DNAthis is used for in situ chromosome mapping using well known techniquesfor this purpose. Typically in accordance with routine procedures forchromosome mapping, some trial and error may be necessary to identify agenomic probe that gives a good in situ hybridization signal.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region of the gene is used to rapidly select primers thatdo not span more than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

PCR mapping of somatic cell-hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with cDNA having at least 50 or60 bases. For a review of this technique, see Verma et al., HumanChromosomes: a Manual of Basic Techniques, Pergamon Press, New York(1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

Comparison of affected and unaffected individuals generally involvesfirst looking for structural alterations in the chromosomes, such asdeletions or translocations that are visible from chromosome spreads ordetectable using PCR based on that cDNA sequence. Ultimately, completesequencing of genes from several individuals is required to confirm thepresence of a mutation and to distinguish mutations from polymorphisms.

Antibodies

Ckβ-6-protein specific antibodies for use in the present invention canbe raised against the intact Ckβ-6 protein or an antigenic polypeptidefragment thereof, which may presented together with a carrier protein,such as an albumin, to an animal system (such as rabbit or mouse) or, ifit is long enough (at least about 25 amino acids), without a carrier.

As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab)is meant to include intact molecules as well as antibody fragments (suchas, for example, Fab and F(ab′)2 fragments) which are capable ofspecifically binding to MPIF-1, M-CIF or MIP4 protein. Fab and F(ab′)2fragments lack the Fc fragment of intact antibody, clear more rapidlyfrom the circulation, and may have less non-specific tissue binding ofan intact antibody (Wahl, et al., J. Nucl. Med. 24:316-325 (1983)).Thus, these fragments are preferred.

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, Nature256:495-497 (1975)), the trioma technique, the human B-cell hybridomatechnique (Kozbor, et al., Immunology Today 4:72 (1983)), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.(1985), pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice may be used to express humanized antibodies to immunogenicpolypeptide products of this invention.

The antibodies of the present invention may be prepared by any of avariety of methods. For example, cells expressing the Ckβ-6 protein oran antigenic fragment thereof can be administered to an animal in orderto induce the production of sera containing polyclonal antibodies. In apreferred method, a preparation of Ckβ-6 protein is prepared andpurified to render it substantially free of natural contaminants. Such apreparation is then introduced into an animal in order to producepolyclonal antisera of greater specific activity.

In the most preferred method, the antibodies of the present inventionare monoclonal antibodies (or Ckβ-6 protein binding fragments thereof).Such monoclonal antibodies can be prepared using hybridoma technology(Kohler, et al., Nature 256:495 (1975); Kohler, et al., Eur. J. Immunol.6:511 (1976); Kohler, et al., Eur. J. Immunol. 6:292 (1976); Hammerling,et al., in Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y.,(1981), pp. 563-681). In general, such procedures involve immunizing ananimal (preferably a mouse) with an Ckβ-6 protein antigen or, morepreferably, with an Ckβ-6 protein-expressing cell. Suitable cells can berecognized by their capacity to bind anti-Ckβ-6 protein antibody. Suchcells may be cultured in any suitable tissue culture medium; however, itis preferable to culture cells in Earle's modified Eagle's mediumsupplemented with 10% fetal bovine serum (inactivated at about 56° C.),and supplemented with about 10 g/l of nonessential amino acids, about1,000 U/ml of penicillin, and about 100 g/ml of streptomycin. Thesplenocytes of such mice are extracted and fused with a suitable myelomacell line. Any suitable myeloma cell line may be employed in accordancewith the present invention; however, it is preferable to employ theparent myeloma cell line (SP20), available from the American TypeCulture Collection, Rockville, Md. After fusion, the resulting hybridomacells are selectively maintained in HAT medium, and then cloned bylimiting dilution as described by Wands et al., Gastroenterology80:225-232 (1981). The hybridoma cells obtained through such a selectionare then assayed to identify clones which secrete antibodies capable ofbinding the Ckβ-6 protein antigen.

Alternatively, additional antibodies capable of binding to the Ckβ-6protein antigen may be produced in a two-step procedure through the useof anti-idiotypic antibodies. Such a method makes use of the fact thatantibodies are themselves antigens, and that, therefore, it is possibleto obtain an antibody which binds to a second antibody. In accordancewith this method, Ckβ-6-protein specific antibodies are used to immunizean animal, preferably a mouse. The splenocytes of such an animal arethen used to produce hybridoma cells, and the hybridoma cells arescreened to identify clones which produce an antibody whose ability tobind to the Ckβ-6 protein-specific antibody can be blocked by the Ckβ-6protein antigen. Such antibodies comprise anti-idiotypic antibodies tothe Ckβ-6 protein-specific antibody and can be used to immunize ananimal to induce formation of further Ckβ-6 protein-specific antibodies.

It will be appreciated that Fab and F(ab′)2 and other fragments of theantibodies of the present invention may be used according to the methodsdisclosed herein. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). Alternatively, Ckβ-6protein-binding fragments can be produced through the application ofrecombinant DNA technology or through synthetic chemistry.

It may be preferable to use “humanized” chimeric monoclonal antibodies.Such antibodies can be produced using genetic constructs derived fromhybridoma cells producing the monoclonal antibodies described above.Methods for producing chimeric antibodies are known in the art. See, forreview, Morrison, Science 229:1202 (1985); Oi, et al., BioTechniques4:214 (1986); Cabilly, et al., U.S. Pat. No. 4,816,567; Taniguchi, etal., EP 171496; Morrison, et al., EP 173494; Neuberger, et al., WO8601533; Robinson, et al., WO 8702671; Boulianne, et al., Nature 312:643(1984); Neuberger, et al., Nature 314:268 (1985).

Further suitable labels for the CkP-6 protein-specific antibodies of thepresent invention are provided below. Examples of suitable enzyme labelsinclude malate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphatedehydrogenase, triose phosphate isomerase, peroxidase, alkalinephosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase, and acetylcholine esterase.

Examples of suitable radioisotopic labels include ³H, ¹¹¹In, ¹²⁵I, ¹³¹I,³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, ⁶⁷Cu, ²¹⁷Ci,²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, etc. ¹¹¹In is a preferred isotope where invivo imaging is used since its avoids the problem of dehalogenation ofthe ¹²⁵I or ¹³⁵I-labeled monoclonal antibody by the liver. In addition,this radionucleotide has a more favorable gamma emission energy forimaging (Perkins, et al., Eur. J. Nucl. Med. 10:296-301 (1985);Carasquillo, et al., J. Nucl. Med. 28:281-287 (1987)).

Examples of suitable non-radioactive isotopic labels include ¹⁵⁷Gd,⁵⁵Mn, ¹⁶² Dy, ⁵²Tr, and ⁵⁶Fe.

Examples of suitable fluorescent labels include an ¹⁵²Eu label, afluorescein label, an isothiocyanate label, a rhodamine label, aphycoeiythrin label, a phycocyanin label, an allophycocyanin label, ano-phthaldehyde label, and a fluorescamine label.

Examples of suitable toxin labels include diphtheria toxin, ricin, andcholera toxin.

Examples of chemiluminescent labels include a luminal label, anisoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridinium salt label, an oxalate ester label, a luciferinlabel, a luciferase label, and an aequorin label.

Examples of nuclear magnetic resonance contrasing agents include heavymetal nuclei such as Gd, Mn, and iron.

Typical techniques for binding the above-described labels to antibodiesare provided by Kennedy, et al., Clin. Chim. Acta 70:1-31 (1976), andSchurs, et al., Clin. Chim. Acta 81:1-40 (1977). Coupling techniquesmentioned in the latter are the glutaraldehyde method, the periodatemethod, the dimaleimide method, them-maleimidobenzyl-N-hydroxy-succinimide ester method, all of whichmethods are incorporated by reference herein.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described.

“Plasmids” are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 mg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 ml of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 mgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.8:4057 (1980).

“Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units to T4 DNA ligase (“ligase”)per 0.5 mg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described inthe method of Graham, F. and Van der Eb, A., Virology 52:456-457 (1973).

EXAMPLE 1 Bacterial Expression and Purification of Ckβ-6

The DNA sequence encoding for Ckβ-6, ATCC # 75703, is initiallyamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ sequences of the processed Ckβ-6 protein (minus the signal peptidesequence) and the vector sequences 3′ to the Ckβ-6 gene. Additionalnucleotides corresponding to Ckβ-6 were added to the 5′ and 3′ sequencesrespectively. The 5′ oligonucleotide primer has the sequence5′TCAGGATCCCCTACGGGCTCGTGGTC 3′ (SEQ ID NO:3) contains a Bam H1restriction enzyme site followed by 18 nucleotides of Ckβ-6 codingsequence starting from the presumed terminal amino acid of the processedprotein codon. The 3′ sequence 3′ CGCTCTAGAGTAAAACGACGGCCAGT 5′ (SEQ IDNO:4) contains complementary sequences to the XbaI site and to apBluescript SK-vector sequence located 3′ to the Ckβ-6 DNA insert. Therestriction enzyme sites correspond to the restriction enzyme sites onthe bacterial expression vector pQE-9. (Qiagen, Inc. 9259 Eton Avenue,Chatsworth, Calif., 91311). pQE-9 encodes antibiotic resistance (Amp′),a bacterial origin of replication (ori), an IPTG-regulatable promoteroperator (P/O), a ribosome binding site (RBS), a 6-His tag andrestriction enzyme sites. pQE-9 was then digested with Bam H1 and Xba I.The amplified sequences were ligated into pQE-9 and were inserted inframe with the sequence encoding for the histidine tag and the RBS. Theligation mixture was then used to transform E. coli strain m15/rep4available from Qiagen under the trademark M15/rep 4 by the proceduredescribed in Sambrook, J. et al, Molecular Cloning: A Laboratory Manual,Cold Spring Laboratory Press (1989). M15/rep4 contains multiple copiesof the plasmid pREP4, which expresses the lacI repressor and alsoconfers kanamycin resistance (Kan′). Transformants are identified bytheir ability to grow on LB plates and ampicillin/kanamycin resistantcolonies were selected. Plasmid DNA was isolated and confirmed byrestriction analysis.

Clones containing the desired constructs were grown overnight (O/N) inliquid culture in LB media supplemented with both Amp (100 μg/ml) andKan (25 μg/ml). The O/N culture is used to inoculate a large culture ata ratio of 1:100 to 1:250. The cells were grown to an optical density600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalactopyranoside”) was then added to a final concentration of 1 mM. IPTGinduces by inactivating the lacI repressor, clearing the P/O leading toincreased gene expression. Cells were grown an extra 3 to 4 hours. Cellswere then harvested by centrifugation. The cell pellet was solubilizedin the chaotropic agent 6 M Guanidine HCl. After clarification,solubilized Ckβ-6 was purified from this solution by chromatography on aNickel-Chelate column under conditions that allow for tight binding byproteins containing the 6-His tag. Hochuli, E. et al., J. Chromatography411:177-184 (1984). Ckβ-6 (95% pure) was eluted from the column in 6 Mguanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3 Mguanidine HCl, 100 mM sodium phosphate, 10 mM glutathione (reduced) and2 mM glutathione (oxidized). After incubation in this solution for 12hours the protein was dialyzed to 10 mM sodium phosphate.

The following preferred alternative method may be used to purify Ckβ-6expressed in E. coli when it is present in the form of inclusion bodies.Unless otherwise specified, all of the following steps are conducted at4-10° C.

Upon completion of the production phase of the E. coli fermentation, thecell culture is cooled to 4-10° C. and the cells are harvested bycontinuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basisof the expected yield of protein per unit weight of cell paste and theamount of purified protein required, an appropriate amount of cellpaste, by weight, is suspended in a buffer solution containing 100 mMTris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneoussuspension using a high shear mixer.

The cells ware then lysed by passing the solution through amicrofluidizer (Microluidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GUHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the Ckβ-6polypeptide-containing supernatant is incubated at 4° C. overnight toallow further GUHCl extraction.

Following high speed centrifugation (30,000×g) to remove insolubleparticles, the GUHCl solubilized protein is refolded by quickly mixingthe GUHCl extract with 20 volumes of buffer containing 50 mM sodium, pH4.5, 150 mMM NaCl, 2 mM EDTA by vigorous stirring. The refolded dilutedprotein solution is kept at 4° C. without mixing for 12 hours prior tofurther purification steps. To clarify the refolded Ckβ-6 polypeptidesolution, a previously prepared tangential filtration unit equipped with0.16 μm membrane filter with appropriate surface area (e.g., Filtron),equilibrated with 40 mM sodium acetate, pH 6.0 is employed. The filteredsample is loaded onto a cation exchange resin (e.g., Poros HS-50,Perseptive Biosystems). The column is washed with 40 mM sodium acetate,pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in thesame buffer, in a stepwise manner. The absorbance at 280 mm of theeffluent is continuously monitored. Fractions are collected and furtheranalyzed by SDS-PAGE.

Fractions containing the Ckβ-6 polypeptide are then pooled and mixedwith 4 volumes of water. The diluted sample is then loaded onto apreviously prepared set of tandem columns of strong anion (Poros HQ-50,Perseptive Biosystems) and weak anion (Poros CM-20, PerseptiveBiosystems) exchange resins. The columns are equilibrated with 40 mMsodium acetate, pH 6.0. Both columns are washed with 40 mM sodiumacetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodiumacetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractionsare collected under constant A₂₈₀ monitoring of the effluent. Fractionscontaining the Ckβ-6 polypeptide (determined, for instance, by 16%SDS-PAGE) are then pooled.

The resultant Ckβ-6 polypeptide exhibits greater than 95% purity afterthe above refolding and purification steps. No major contaminant bandsare observed from Commassie blue stained 16% SDS-PAGE gel when 5 μg ofpurified protein is loaded. The purified protein is also tested forendotoxin/LPS contamination, and typically the LPS content is less than0.1 ng/ml according to LAL assays.

EXAMPLE 2 Expression Pattern of Ckβ-6 in Human Cells

Northern blot analysis was carried out to examine the levels ofexpression of Ckβ-6 in human cells. Total cellular RNA samples wereisolated with RNAzol′ B system (Biotecx Laboratories, Inc. 6023 SouthLoop East, Houston, Tex. 77033). About 10 μg of total RNA isolated fromeach human tissue specified was separated on 1% agarose gel and blottedonto a nylon filter. (Sambrook, Fritsch, and Maniatis, MolecularCloning, Cold Spring Harbor Press (1989)). The labeling reaction wasdone according to the Stratagene Prime-It kit with 50 ng DNA fragment.The labeled DNA was purified with a Select-G-50 column. (5 Prime-3Prime, Inc. 5603 Arapahoe Road, Boulder, Colo. 80303). The filter wasthen hybridized with radioactive labeled full length Ckβ-6 gene at1,000,000 cpm/ml in 0.5 M NaPO₄, pH 7.4 and 7% SDS overnight at 65° C.After wash twice at room temperature and twice at 60° C. with 0.5×SSC,0.1% SDS, the filter was then exposed at −70° C. overnight with anintensifying screen. The message RNA for Ckβ-6 is abundant in activatedand unactivated T cells, monocytes and T cell lines.

EXAMPLE 3 Cloning and Expression of Ckβ-6 Using the BaculovirusExpression System

The DNA sequence encoding the full length Ckβ-6 protein, ATCC # 75703,is amplified using PCR oligonucleotide primers corresponding to the 5′and 3′ sequences of the gene:

The amplified sequences were isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment was then digested with restriction endonucleasescorresponding to the amplified products and then purified again on a 1%agarose gel. This fragment is designated F2.

The vector pRG1 (modification of pVL941 vector, discussed below) is usedfor the expression of the Ckβ-6 protein using the baculovirus expressionsystem (for review see: Summers, M. D. and Smith, G. E. A manual ofmethods for baculovirus vectors and insect cell culture procedures,Texas Agricultural Experimental Station Bulletin No. 1555 (1987)). Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed bythe recognition sites for the restriction endonucleases used to digestthe amplified products. The polyadenylation site of the simian virus(SV)40 is used for efficient polyadenylation. For an easy selection ofrecombinant virus the beta-galactosidase gene from E. coli is insertedin the same orientation as the polyhedrin promoter followed by thepolyadenylation signal of the polyhedrin gene. The polyhedrin sequencesare flanked at both sides by viral sequences for the cell-mediatedhomologous recombination of co-transfected wild-type viral DNA. Manyother baculovirus vectors could be used in place of pRG1 such as pAc373,pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

The plasmid is digested with the restriction enzymes anddephosphorylated using calf intestinal phosphatase by procedures knownin the art. The DNA was then isolated from a 1% agarose gel using thecommercially available kit (“Geneclean” BIO 101 Inc., La Jolla, Calif.).This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNAligase. E. coli HB101 cells are then transformed and bacteria identifiedthat contained the plasmid (pBacCkβ-6) with the Ckβ-6 gene using theenzymes. The sequence of the cloned fragment is confirmed by DNAsequencing.

5 mg of the plasmid pBacCkβ13-6 is co-transfected with 1.0 mg of acommercially available linearized baculovirus (“BaculoGold baculovirusDNA”, Pharmingen, San Diego, Calif.) using the lipofection method(Felgner, et al., Proc. Natl. Acad. Sci. (USA) 84:7413-7417 (1987)).

1 mg of BaculoGold virus DNA and 5 mg of the plasmid pBacCkβ-6 are mixedin a sterile well of a microtiter plate containing 50 ml of serum freeGrace's medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards10 ml Lipofectin plus 90 ml Grace's medium are added, mixed andincubated for 15 minutes at room temperature. Then the transfectionmixture is added drop-wise to the Sf9 insect cells (ATCC CRL 1711)seeded in a 35 mm tissue culture plate with 1 ml Grace's medium withoutserum. The plate is rocked back and forth to mix the newly addedsolution. The plate is then incubated for 5 hours at 27° C. After 5hours the transfection solution is removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum is added.The plate was put back into an incubator and cultivation continued at27° C. for four days.

After four days the supernatant is collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) is used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

Four days after the serial dilution, the virus is added to the cells,blue stained plaques are picked with the tip of an Eppendorf pipette.The agar containing the recombinant viruses is then resuspended in anEppendorf tube containing 200 ml of Grace's medium. The agar is removedby a brief centrifugation and the supernatant containing the recombinantbaculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Fourdays later the supernatants of these culture dishes are harvested andthen stored at 4° C.

Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-Ckβ-6 at a multiplicity of infection (MOI) of 2. Six hourslater the medium is removed and replaced with SF900 II medium minusmethionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hourslater 5 mCi of ³⁵S-methionine and 5 mCi ³⁵S cysteine (Amersham) areadded. The cells are further incubated for 16 hours before they areharvested by centrifugation and the labelled proteins visualized bySDS-PAGE and autoradiography.

Ckβ-6 produced essentially according to the above procedure was purifiedfrom the serum free insect cell supernatent by cation exchange, heparinaffinity, and size exclusion chromatography (poros 50 HS, poros 20 HE1,Perseptive Biosystem, and Sephacryl S200 HR; Pharmacia) in the presenceof protease inhibitors (20 mg/ml Pefabloc SC; Boehringer Mannheim, 1mg/ml leupeptin, 1 mg/ml E64, and 1 mM EDTA).

Analysis of the purified protein was performed by laser desorption massspectrometry (matrix-associated laser desportion ionization-time offlight) and by Edman degradation after partial proteolysis withendoproteinase GluC (Boehringer Mannheim).

EXAMPLE 4 Expression via Gene Therapy

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T., et al, DNA 7:219-25 (1988)) flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention is amplifiedusing PCR primers which correspond to the 5′ and 3′ end sequencesrespectively. The 5′ primer contains an EcoRI site and the 3′ primerfurther includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HB101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interestproperly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellsare transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product.

EXAMPLE 5 Primary Indication of Ckβ-6 as a Mobilizer of Marrow StemCells (Bone Marrow Rescue)

The effect of Ckβ-6 on the distribution of the primitive hematopoieticprogenitors in peripheral blood, spleen, and bone marrow was studied in16 week old C57B1/6 mice (about 20 g). In the first experiment, 3 micewere injected i.p. daily with 1 mg/kg Ckβ-6 or saline for 2 days andanalyzed 24 hours after the last injection. In the second experiment,another 3 mice were injected i.p. daily with 1 mg/kg Ckβ-6 or saline for4 days and analyzed 24 hours after the last injection. In both theexperiments, the blood of each animal was collected by cardiac punctureand the mice were sacrificed to obtain bone marrow and spleens. Theindicated number of cells from each of the tissues was then plated induplicates in agar-containing medium in the presence of 5 ng/ml IL-3, 50ng/ml SCF, 5 ng/ml M-CSF and 10 ng/ml IL-1a and incubated for 14 days.In the 2 experiments, the data from the different animals were pooledand expressed as mean±S.D. The results of both experiments shows thatCkβ-6 mobilize stem cells from bone marrow to peripheral blood (Tables 2and 3). In the first experiment, after 2 days of treatment with Ckβ-6,the frequency of HPP-CFC, LPP-CFC and immature cells in peripheral bloodincreased significantly over the controls. No changes were observed inthe spleen and a significant decrement of HPP-CFC was observed in thebone marrow (Table 3). In the second experiment, after 4 days oftreatment with Ckβ-6, the same significant increment of HPP-CFC, LPP-CFCand immature cells frequency was observed in peripheral blood. Asignificant increment of immature cells frequency was observed in thespleen and a significant decrement of HPP-CFC and LPP-CFC was observedin the bone marrow (Table 3). In particular it is important to note thepresence of immature hematopoietic cells in the peripheral blood afterthe injection of Ckβ-6. The effect was observed in the animals treatedwith Ckβ-6 was not due to toxicity as the FACScan profile of theleukocyte composition of both the control and the mice treated withCkβ-6 is identical (Table 4).

EXAMPLE 6 Ckβ-6 as a Myeloprotectant Against Cytosine Arabinoside

In this experiment, Lin− cells were plated (1×10⁵ cell/ml) in a growthmedium that was supplemented with 5 ng/ml mouse IL-3, 50 ng/ml mouse SCF(column 1); IL-3, SCF and 100 ng/ml Ckβ-6 (column 2); or IL-3, SCF and100 ng/ml of the irrelevant protein HG200-3-B (column 3). After 48 hoursof incubation, one set of the above cultures received 50 mg/ml Ara-C andthe incubation was then continued for an additional 24 hours. Cells werethen harvested, washed three times with HBSS to remove the drug and thecytokines, and assayed for the presence of HPP-CFC and LPP-CFC asdescribed in the legend to FIG. 4. The results are expressed as mean %of protection (±SD). The % of protection was calculated as follows:Percent protection is expressed as number of colonies found in culturesincubated in the presence of Ara-C divided by the number of coloniesfound in cultures incubated without Ara-C×100. Data from one out of 3experiments are shown in FIG. 6. All the samples were tested induplicates.

EXAMPLE 7 Ckβ-6 as a Myeloprotectant Against 5-Fluorouracil

Mononuclear population of mouse bone marrow cells was depleted oflineage-committed cells by negative selection using a panel monoclonalantibodies directed against cell surface antigens. The resultingpopulation of cells (Lin.− cells) were resuspended (1×10⁵ cells/ml) in agrowth medium containing IL-3 (5 ng/ml), SCF (50 ng/ml), GM-CSF (5ng/ml), M-CSF (5 ng/ml) and IL-1a (10 ng/ml) and 1 ml of this cellsuspension was dispensed into culture tubes. (1) A set of duplicatecultures received no chemoline; (2) duplicate cultures with Ckβ-6 at 100ng/ml; and (3) duplicate cultures with an irrelevant protein at 100ng/ml. All cultures were incubated in a tissue culture incubator for 48hours, at which point one culture from each set received 5-Fluorouracilat 100 mg/ml and incubation was continued for additional 24 hours. Allcultures were then harvested, washed three times with HBSS, and thenassayed for the presence of the HPP-CFC & LPP-CFC as described in thelegend to FIG. 5. Percent protection is expressed as number of coloniesdetected in cultures incubated in the presence of 5-FU divided by thenumber of colonies found in cultures incubated without 5-FU×100. Dataare expressed as Mean±SD. Two experiments were performed and each assaywas in duplicates. See FIG. 7.

EXAMPLE 8 Ckβ-6 Effect on Cortical Neuronal Survival

Sprague-Dawley rats at gestation day 17 were sacrificed and the cortexwas removed and the meninges were carefully pealed away from thecortical tissue pieces. Single cell suspensions were prepared and thecells were plated in medium containing 5% horse serum at a density of20,000 cells/well. After 24 hours the serum containing medium wasremoved and serum-free medium was added to the cultures. Included in theserum-free cultures was a concentration of Ckβ-6 as shown in FIG. 8. TheCkβ-6 used is an Ckβ-6 polypeptide encoded by the polynucleotidesequence as shown in SEQ ID NO:1 of the application. The medium waschanged every other day and Ckβ-6 was added again. The neurons weremaintained in culture for 6 days prior to the viability assay.

Cell viability was assessed using the live/dead assay kit from MolecularProbes. This assay is a two-color fluorescence cell viability assaybased on the simultaneous determination of live and dead cells. Livecells are distinguished by the presence of ubiquitous intracellularesterase activity, determined by enzymatic conversion of the nearlynon-fluorescent cell permeant calcein AM to the intensely fluorescentcalcein. The polycationic calcein is well retained by living cells andthus produces an intense uniform green fluorescence in living cells.Thus the emission reading (approximately 530 nm) is a measurement of thetotal cell number of the cultures. As shown in FIG. 8, the number oflive cells increased as the concentration of Ckβ-6 increased.

EXAMPLE 9 Leukocyte Response and Receptor Usage

Monocytes, lymphocytes, and neutrophils were isolated from donor bloodbuffy coats. Eosinophils and basophils were purified from fresh venousblood of healthy volunteers.

Changes in the cytosolic free Ca²⁺ concentration ([Ca²⁺]i), and enzymerelease were monitored in monocytes, eosinophils, lymphocytes andneutrophils loaded with Fura-2 acetoxymethyl ester (0.2 nmol per 10⁶cells) by incubation for 20 min. at 37° C. in medium containing 136 mMNaCl, 4.8 mM Kcl, 1 mM CaCl₂, 5 mM glucose, and 20 mM Hepes, pH 7.4 and1 to 1,000 nM Ckβ-6 in comparison with IL-8, MCP-1 and MCP-3. Loadedcells were washed and resuspended in the same medium (10⁶ cells/ml) and[Ca²⁺]i-related fluorescence changes were recorded. Receptordesensitization was tested by monitoring [Ca²⁺]i changes aftersequential chemokine stimulation.

In three independent experiments no effects of Ckβ-6 were observed onneutrophils, monocytes and T-lymphocytes, but considerable activity wasfound on eosinophils. In these cells, cross-desensitization betweenCkβ-6 on the one ahand and eotaxin, MCP-3, RANTES or MIP-1α on the otherwas studied by monitoring [Ca²⁺]i changes. As shown in FIG. 9,stimulation of eosinophils with Ckβ-6 abrogated the response to eotaxin,attenuated the responses to MCP-3 and RANTES, but did not appreciablyaffect the response to MIP-1α. In agreement with these results the[Ca²⁺]i rise induced by Ckβ-6 was abrogated by prior stimulation witheotaxin, decreased by stimulation with RANTES or MCP-3, but not affectedby MIP-1α. The complete cross-desensitization with eotaxin suggests thatCkβ-6 also acts via CCR3.

EXAMPLE 10 In-vitro Chemotaxis

Chemotaxis was assessed in 48-well chambers (Neuro Probe, Cabin John,Md.) using polyvinylpyrrolidone-free polycarbonate membranes(Nucleopore) with 5-μm pores for eosinophils and basophils, and 3-μmpores for lymphocytes. RPMI 1640 supplemented with 20 mM Hepes, pH 7.4,and 1% pasteurized plasma protein solution (the Central Laboratory ofthe Swiss Red Cross) was used for the cell suspensions and chemokinedilutions. After an incubation of 60 min. at 37° C. in 5% CO2, themembrane was removed, washed on the upper side with PBS, fixed, andstained. All assays were done in triplicate, and the migrated cells werecounted in five randomly selected fields at 1,000-fold magnification.Spontaneous migration was determined in the absence of chemoattractant.

Results. In vitro chemotaxis was tested with human blood monocytes, Tlymphocytes, eosinophil and basophil leukocytes. No activity was foundtoward monocytes and lymphocytes, which agrees with the lack of [Ca²⁺]ichanges, but marked migration was obtained with eosinophils andbasophils. As shown in FIG. 10, Ckβ-6 is a very effective attractant forboth types of cells. When the assay was performed in the presence ofanti-CCR3, eosinophil and basophil chemotaxis toward Ckβ-6 wascompletely prevented.

EXAMPLE 11 Histamine and Leukotriene C₄ (LTC4) Release

Basophils (0.1 to 0.3×10⁶ cells/ml) in 20 mM Hepes, pH 7.4 containing125 mM glucose and 0.025% BSA were warmed to 37° C., exposed to IL-3 (10ng/ml) with or without anti-CCR3 (10 μg/ml) and then challenged with achemokine. After 20 min. the reaction was stopped by placing the tubeson ice and histamine and LTC₄ were measured in the supernatant.Histamine release was expressed as percent of the total content of thesample (determined after cell lysis).

LTC₄ generation was expressed in nanogram per 10⁶ basophils, as shown inFIG. 11. On IL-3 pretreated basophils from several unselected donorsboth Ckβ-6 and eotaxin induced similar release of histamine and peptidoleukotrines at maximum effective concentrations. The curves relatingeffect to concentration shown that eotaxin was slightly more potent thanCkβ-6 in particular as inducer of LTC₄ release. In both assays, Ckβ-6was approximately as potent as RANTES and MIP-1α (data not shown). Asexpected, in consideration of effects on chemotaxis, above, the releaseresponses to Ckβ-6 was markedly inhibited by pretreatment withanti-CCR3.

EXAMPLE 12 In-vivo Activity

Since human eotaxin is active in monkeys and induces the localaccumulation of eosinophils after intradermal injection, its effect inrhesus monkey was compared with Ckβ-6.

A male rhesus monkey of 7.5 kg was anesthetized by i.m. injection of 10mg/kg Ketamine (Ketolar, Parke Davis) and i.v. injection of 15 mg/kg NaThiopental (Pentotal, Abbott). Chemokines in 100 μl pyrogen-freeisotonic saline (100 pmol eotaxin, 100 and 1,000 pmol Ckβ-6) were theninjected intradermally on the back and full skin thickness punchbiopsies of 8 mm diameter were taken from the injection sites after 4 h.The biopsies were fixed in formalin, embedded in parafin, and 5 μmsections were prepared. The sections were stained with hematoxylin andeosin, and the infiltrates were evaluated by two independent observers.Eosinophil counting was performed at a magnification of 630× in fiverandomly selected fields per section next to and including apostcapillary venule of the superficial vascular plexus using a countinggrid of 0.19×0.19 mm, and the number of eosinophils per mm² wascalculated.

As shown in FIG. 12, 4 hours after injection of 100 pmol per site bothchemokines induced a similar, marked eosinophil infiltration as comparedto the vehilce alone, which had no effect. A 50% higher number ofinfiltrating eosinophils was counted when 1000 pmol Ckβ-6 were applied.The effect was remarkable because the monkey used in this experiment hada low blood eosinophil count (0.7% of total leukocytes).

EXAMPLE 13 Ckβ-6 Agonists and Antagonists

Several deletion Ckβ-6 mutants were constructed and assayed for activityusing a [Ca+²]i flux assay and a chemotaxis assay. These mutants wereconstructed based upon a Ckβ-6 nucleotide sequence wherein the codonswere optimized for expression in E. coli. The constructs were made asfollows:

Codon Optimized Construct for Ckβ-6 Expression in E. coli

The initial PCR was done using primers 1, 2, 3 and 4 (below) and furtheramplified with primers 5 and 6 (below). The product was digested withNde I and Asp I and cloned into pHE4 for E. coli expression. (See, FIG.21). The resulting codon optimized sequence for expression of Ckβ-6 isshown in SEQ ID NO:6.

Primer #1:

5′ GAC TCC ATG GTG GTT ATA CCT TCT CCG TGC TGC ATG TTC TTT GTT AGC AAGCGC ATT CCT GAA AAC CGT GTG GTC A GCT ACC AGC TGT CCA GCC GC 3′(SEQ IDNO:7).

Primer #2:

5′ GTT TCG GGT CGC CAC AGA ACT GCT GGC CCT TTT TGG TGG TGA AGA TCA CGCCAG CTT TCA GGC AGG TGC TGC GGC TGG ACA GCT GGT AGC TGA CCA C 3′(SEQ IDNO:8).

Primer #3:

5′ AAG GGC CAG CAG TTC TGT GGC GAC CCG AAA CAA GAG TGG GTC CAG CGT TACATG AAA AAC CTG GAC GCC AAA CAG AAG AAA GCT TCC CCT CGT GCC CG 3′ (SEQID NO:9)

Primer #4:

5′ AGT CAG ATC TTT AGC AGG TGG TTT GGT TGC CCG GAT AAC GCT GAA CAG GGCCTT TGA CAG CCA CTG CGC GGG CAC GAG GGG AAG CTT TCT TCT GTT TGG 3′ (SEQID NO:10).

Primer #5:

5′ GAC GGAT CCC CAT ATG GTG GTT ATA CCT TCT CCG 3′(SEQ ID NO:11). (NdeIsite in bold)

Primer #6:

5′ GAC TGG TAC CTT AGC AGG TGG TTT GGT TGC CC 3′ (SEQ ID NO:12). (Asp 1site in bold)

Using this E. coli optimized codon Ckβ-6 construct, the followingdeletion mutants, which include a methionine at the N-terminus, weregenerated using the primers listed below:

ΔC1 amino acids 1 to 73 in SEQ ID NO:2 ΔC1ΔN1 amino acids 2 to 73 in SEQID NO:2 ΔC1ΔN2 amino acids 3 to 73 in SEQ ID NO:2 ΔC1ΔN3 amino acids 4to 73 in SEQ ID NO:2 ΔC1ΔN4 amino acids 5 to 73 in SEQ ID NO:2 ΔC1ΔN5amino acids 6 to 73 in SEQ ID NO:2 ΔC1ΔN6 amino acids 7 to 73 in SEQ IDNO:2

ΔC1:

5′ primer: 5′ GACGGATCCCCATATGGTGGTTATACCTTCTCCG 3′ (SEQ ID NO:11).

3′ primer: 5′ GACTGGTACCTTATCAACGAGGGGAAGCTTTCTTCT 3′(SEQ ID NO:13).

ΔC2:

5′ primer: 5′ GACGGATCCCCATATGGTGGTTATACCTTCTCCG 3′ (SEQ ID NO:11).

3′ primer: 5′ GACTGGTACCCTATCAAGCCACTGCGCGGGCACGAGG 3′ (SEQ ID NO:14).

ΔC1ΔN1:

5′ primer: 5′ GACTCATATGGTTATACCTTCTCCGTGCTGCATG 3′ (SEQ ID NO:15).

3′ primer: 5′ GACTGGTACCTTATCAACGAGGGGAAGCTTTCTTCT 3′ (SEQ ID NO:13).

ΔC1ΔN2:

5′ primer: 5′ GACTCATATGATACCTTCTCCGTGCTGCATG 3′ (SEQ ID NO:16).

3′ primer: 5′ GACTGGTACCTTATCAACGAGGGGAAGCTTTCTTCT 3′ (SEQ ID NO:13).

ΔC1ΔN3:

5′ primer: 5′ GACTCATATGCCTTCTCCGTGCTGCATGTTC 3′ (SEQ ID NO:17).

3′ primer: 5′ GACTGGTACCTTATCAACGAGGGGAAGCTTTCTTCT 3′ (SEQ ID NO:13).

ΔC1ΔN4:

5′ primer: 5′ GACTCATATGTCTCCGTGCTGCATGTTCTTTG 3′ (SEQ ID NO:18).

3′ primer: 5′ GACTGGTACCTTATCAACGAGGGGAAGCTTTCTTCT 3′ (SEQ ID NO:13).

ΔC1ΔN5:

5′ primer: 5′ GACTCATATGCCGTGCTGCATGTTCTTTG 3′ (SEQ ID NO:19).

3′ primer: 5′ GACTGGTACCTTATCAACGAGGGGAAGCTTCTCT 3′ (SEQ ID NO:13).

ΔC1ΔN6:

5′ primer: 5′ GACTCATATGTGCTGCATGTTCTTTGTTAG 3′ (SEQ ID NO:20).

3′ primer: 5′ GACTGGTACCTTATCAACGAGGGGAAGCTTTCTTCT 3′ (SEQ ID NO:13).

Several of these deletion Ckβ-6 mutants were assayed for activity usinga [Ca+²]i flux assay and a chemotaxis assay.

Calcium Flux Assay

The calcium flux assay was performed using eosinophils essentially asdescribed in Example 9, above. It should be noted that for the proteinpreparations assayed, HG00603 and HG00605 retained the N-terminalmethionine; HG00606, HG00608 and HG00609 did not have the N-terminalmethionine; and in HG00604, about 55% of the protein had the N-terminalmethionine.

Results

Four of these mutants (ΔC1ΔN1, ΔC1ΔN2, ΔC1ΔN3, ΔC1ΔN5) were used incalcium flux assays with primary Eosinopils. ΔC1ΔN1 (HG00604) and ΔC1ΔN2(HG00605) showed activities which were very similar to ΔC1 (HG00603) andEotaxin whereas ΔC1ΔN3 (HG00606) and ΔC1ΔN5 (HG00608) showed no activityin this assay even at 1000 ng/ml (see, FIGS. 14A and 14B).

When combined with ΔC1N1 (HG00604), ΔC1ΔN3 (HG00606) was able to inhibitthe activity of ΔC1ΔN1 (HG00604) whereas ΔC1ΔN5 (HG00608) was not (seeFIGS. 15A and 15B). In addition, when combined with ΔC1ΔN1, Ckβ-10 orEotaxin, ΔC1ΔN3 (HG00606) was able to inhibit the activity of all threeof these chemokines (see FIGS. 16A and 16B). ΔC1ΔN3 (HG00606) is anefficient antagonist of all three of these chemokines due to the factthat these chemokines have all been shown to signal through the samereceptor, CCR3.

In Vitro Chemotaxis Assay

Cells were washed and labeled with calcein-AM and distributed into theupper chamber of a 96 well disposable chemotaxis plate (NeuroProbe,Cabin John, Md.) separated by a polycarbonate filter (5-8 μm pore size;PVP free; NeuroProbe, Inc.) Cells were allowed to migrate for 90 minutes(lymphocytes) or 3 hours (eosinophils) and then the number of migratedcells (both attached to the filter as well as in the bottom chamber)were quantitated using a Cytofluor II fluorescence plate reader(PerSeptive Biosystems). Values for the chemotaxis assay are reported asthe chemotatic index which refers to the fold induction above backgroundobserved with the various factors used. It should be noted that, for theprotein preparations assayed, HG00603 and HG00605 retained theN-terminal methionine; HG00606, HG00608 and HG00609 did not retain theN-terminal methionine; and in HG00604, about 55% of the protein retainedthe N-terminal methionine.

Results

Chemotaxis assays using mutant ΔC1ΔN3 (HG00606) show that this truncatedprotein is no longer active (FIG. 17). To determine if this proteincould serve as an antagonist of eosinophil chemotaxis, experiments wereperformed with ΔC1 (HG00603), Ckβ-10 or Eotaxin with and without ΔC1ΔN3(HG00606). ΔC1ΔN3 (HG00606) (1000 ng/ml) was added to both the top andbottom wells of the chemotaxis chambers with increasing amounts of theother chemokines in the bottomi well. As shown in FIGS. 18A and B,ΔC1ΔN3 (HG00606) was able to inhibit the chemotaxis of eosinophilsdirected by ΔC1 (HG00603). In addition, ΔC1ΔN3 (HG00606) was able toinhibit both Eotaxin (FIGS. 19A and B) and Ckβ-10 (FIGS. 20A and B)driven chemotaxis. Since all of these chemokines mediate their effectson eosinophils via the CCR3 receptor, ΔC1ΔN3 (HG00606) represents adominant antagonist which is capable of inhibiting the signallingthrough this receptor regardless of the chemokine mediating this effect.

CONCLUSIONS

These results indicate that ΔC1ΔN3 is an antagonists of the CCR3receptor-mediated signalling pathway and is useful for the treatment ofany condition due to the activation of eosinophils or basophils. Thisincludes most pro-inflammatory conditions (both acute and chronic) aswell as allergic responses including asthma, airway inflammation, adultrespiratory distress syndrome and allergies in general. This furtherincludes any disease state due to the over-expression of Ckβ-6, Ckβ-10or Eotaxin since ΔC1ΔN3 inhibits the activity of all these chemokines.In addition, ΔC1ΔN3 can also be used to treat conditions resulting fromthe over-expression and/or over activation of the CCR3 receptor.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference.

114 360 base pairs nucleic acid single linear DNA (genomic) CDS 1..357sig_peptide 1..79 mat_peptide 79..357 1 ATG GCA GGC CTG ATG ACC ATA GTAACC AGC CTT CTG TTC CTT GGT GTC 48 Met Ala Gly Leu Met Thr Ile Val ThrSer Leu Leu Phe Leu Gly Val -26 -25 -20 -15 TGT GCC CAC CAC ATC ATC CCTACG GGC TCT GTG GTC ATA CCC TCT CCC 96 Cys Ala His His Ile Ile Pro ThrGly Ser Val Val Ile Pro Ser Pro -10 -5 1 5 TGC TGC ATG TTC TTT GTT TCCAAG AGA ATT CCT GAG AAC CGA GTG GTC 144 Cys Cys Met Phe Phe Val Ser LysArg Ile Pro Glu Asn Arg Val Val 10 15 20 AGC TAC CAG CTG TCC AGC AGG AGCACA TGC CTC AAG GCA GGA GTG ATC 192 Ser Tyr Gln Leu Ser Ser Arg Ser ThrCys Leu Lys Ala Gly Val Ile 25 30 35 TTC ACC ACC AAG AAG GGC CAG CAG TTCTGT GGC GAC CCC AAG CAG GAG 240 Phe Thr Thr Lys Lys Gly Gln Gln Phe CysGly Asp Pro Lys Gln Glu 40 45 50 TGG GTC CAG AGG TAC ATG AAG AAC CTG GACGCC AAG CAG AAG AAG GCT 288 Trp Val Gln Arg Tyr Met Lys Asn Leu Asp AlaLys Gln Lys Lys Ala 55 60 65 70 TCC CCT AGG GCC AGG GCA GTG GCT GTC AAGGGC CCT GTC CAG AGA TAT 336 Ser Pro Arg Ala Arg Ala Val Ala Val Lys GlyPro Val Gln Arg Tyr 75 80 85 CCT GGC AAC CAA ACC ACC TGC TAA 360 Pro GlyAsn Gln Thr Thr Cys 90 119 amino acids amino acid linear protein 2 MetAla Gly Leu Met Thr Ile Val Thr Ser Leu Leu Phe Leu Gly Val -26 -25 -20-15 Cys Ala His His Ile Ile Pro Thr Gly Ser Val Val Ile Pro Ser Pro -10-5 1 5 Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu Asn Arg Val Val10 15 20 Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys Ala Gly Val Ile25 30 35 Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp Pro Lys Gln Glu40 45 50 Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys Gln Lys Lys Ala55 60 65 70 Ser Pro Arg Ala Arg Ala Val Ala Val Lys Gly Pro Val Gln ArgTyr 75 80 85 Pro Gly Asn Gln Thr Thr Cys 90 26 base pairs nucleic aciddouble linear DNA (genomic) 3 TCAGGATCCC CTACGGGCTC GTGGTC 26 26 basepairs nucleic acid double linear DNA (genomic) 4 TGACCGGCAG CAAAATGAGATCTCGC 26 99 amino acids amino acid linear protein 5 Met Lys Val Ser AlaAla Leu Leu Cys Leu Leu Leu Ile Ala Ala Thr 1 5 10 15 Phe Ile Pro GlnGly Leu Ala Gln Pro Asp Ala Ile Asn Ala Pro Val 20 25 30 Thr Cys Cys TyrAsn Phe Thr Asn Arg Lys Ile Ser Val Gln Arg Leu 35 40 45 Ala Ser Tyr ArgArg Ile Thr Ser Ser Lys Cys Pro Lys Glu Ala Val 50 55 60 Ile Phe Lys ThrIle Val Ala Lys Glu Ile Cys Ala Asp Pro Lys Gln 65 70 75 80 Lys Trp ValGln Asp Ser Met Asp His Leu Asp Lys Gln Thr Gln Thr 85 90 95 Pro Lys Thr285 base pairs nucleic acid double linear cDNA 6 ATGGTGGTTA TACCTTCTCCGTGCTGCATG TTCTTTGTTA GCAAGCGCAT TCCTGAAAAC 60 CGTGTGGTCA GCTACCAGCTGTCCAGCCGC AGCACCTGCC TGAAAGCTGG CGTGATCTTC 120 ACCACCAAAA AGGGCCAGCAGTTCTGTGGC GACCCGAAAC AAGAGTGGGT CCAGCGTTAC 180 ATGAAAAACC TGGACGCCAAACAGAAGAAA GCTTCCCCTC GTGCCCGCGC AGTGGCTGTC 240 AAAGGCCCTG TTCAGCGTTATCCGGGCAAC CAAACCACCT GCTAA 285 96 base pairs nucleic acid single linearcDNA 7 GACTCCATGG TGGTTATACC TTCTCCGTGC TGCATGTTCT TTGTTAGCAA GCGCATTCCT60 GAAAACCGTG TGGTCAGCTA CCAGCTGTCC AGCCGC 96 97 base pairs nucleic acidsingle linear cDNA 8 GTTTCGGGTC GCCACAGAAC TGCTGGCCCT TTTTGGTGGTGAAGATCACG CCAGCTTTCA 60 GGCAGGTGCT GCGGCTGGAC AGCTGGTAGC TGACCAC 97 98base pairs nucleic acid single linear cDNA 9 AAGGGCCAGC AGTTCTGTGGCGACCCGAAA CAAGAGTGGG TCCAGCGTTA CATGAAAAAC 60 CTGGACGCCA AACAGAAGAAAGCTTCCCCT CGTGCCCG 98 99 base pairs nucleic acid single linear cDNA 10AGTCAGATCT TTAGCAGGTG GTTTGGTTGC CCGGATAACG CTGAACAGGG CCTTTGACAG 60CCACTGCGCG GGCACGAGGG GAAGCTTTCT TCTGTTTGG 99 34 base pairs nucleic acidsingle linear cDNA 11 GACGGATCCC CATATGGTGG TTATACCTTC TCCG 34 32 basepairs nucleic acid single linear cDNA 12 GACTGGTACC TTAGCAGGTGGTTTGGTTGC CC 32 36 base pairs nucleic acid single linear cDNA 13GACTGGTACC TTATCAACGA GGGGAAGCTT TCTTCT 36 37 base pairs nucleic acidsingle linear cDNA 14 GACTGGTACC CTATCAAGCC ACTGCGCGGG CACGAGG 37 34base pairs nucleic acid single linear cDNA 15 GACTCATATG GTTATACCTTCTCCGTGCTG CATG 34 31 base pairs nucleic acid single linear cDNA 16GACTCATATG ATACCTTCTC CGTGCTGCAT G 31 31 base pairs nucleic acid singlelinear cDNA 17 GACTCATATG CCTTCTCCGT GCTGCATGTT C 31 32 base pairsnucleic acid single linear cDNA 18 GACTCATATG TCTCCGTGCT GCATGTTCTT TG32 29 base pairs nucleic acid single linear cDNA 19 GACTCATATGCCGTGCTGCA TGTTCTTTG 29 30 base pairs nucleic acid single linear cDNA 20GACTCATATG TGCTGCATGT TCTTTGTTAG 30 4256 base pairs nucleic acid doublelinear DNA (genomic) 21 AAGCTTAAAA AACTGCAAAA AATAGTTTGA CTTGTGAGCGGATAACAATT AAGATGTACC 60 CAATTGTGAG CGGATAACAA TTTCACACAT TAAAGAGGAGAAATTACATA TGGTGGTTAT 120 ACCTTCTCCG TGCTGCATGT TCTTTGTTAG CAAGCGCATTCCTGAAAACC GTGTGGTCAG 180 CTACCAGCTG TCCAGCCGCA GCACCTGCCT GAAAGCTGGCGTGATCTTCA CCACCAAAAA 240 GGGCCAGCAG TTCTGTGGCG ACCCGAAACA AGAGTGGGTCCAGCGTTACA TGAAAAACCT 300 GGACGCCAAA CAGAAGAAAG CTTCCCCTCG TGCCCGCGCAGTGGCTGTCA AAGGCCCTGT 360 TCAGCGTTAT CCGGGCAACC AAACCACCTG CTAAGGTACCTAAGTGAGTA GGGCGTCCGA 420 TCGACGGACG CCTTTTTTTT GAATTCGTAA TCATGGTCATAGCTGTTTCC TGTGTGAAAT 480 TGTTATCCGC TCACAATTCC ACACAACATA CGAGCCGGAAGCATAAAGTG TAAAGCCTGG 540 GGTGCCTAAT GAGTGAGCTA ACTCACATTA ATTGCGTTGCGCTCACTGCC CGCTTTCCAG 600 TCGGGAAACC TGTCGTGCCA GCTGCATTAA TGAATCGGCCAACGCGCGGG GAGAGGCGGT 660 TTGCGTATTG GGCGCTCTTC CGCTTCCTCG CTCACTGACTCGCTGCGCTC GGTCGTTCGG 720 CTGCGGCGAG CGGTATCAGC TCACTCAAAG GCGGTAATACGGTTATCCAC AGAATCAGGG 780 GATAACGCAG GAAAGAACAT GTGAGCAAAA GGCCAGCAAAAGGCCAGGAA CCGTAAAAAG 840 GCCGCGTTGC TGGCGTTTTT CCATAGGCTC CGCCCCCCTGACGAGCATCA CAAAAATCGA 900 CGCTCAAGTC AGAGGTGGCG AAACCCGACA GGACTATAAAGATACCAGGC GTTTCCCCCT 960 GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG ACCCTGCCGCTTACCGGATA CCTGTCCGCC 1020 TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT CATAGCTCACGCTGTAGGTA TCTCAGTTCG 1080 GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT GTGCACGAACCCCCCGTTCA GCCCGACCGC 1140 TGCGCCTTAT CCGGTAACTA TCGTCTTGAG TCCAACCCGGTAAGACACGA CTTATCGCCA 1200 CTGGCAGCAG CCACTGGTAA CAGGATTAGC AGAGCGAGGTATGTAGGCGG TGCTACAGAG 1260 TTCTTGAAGT GGTGGCCTAA CTACGGCTAC ACTAGAAGAACAGTATTTGG TATCTGCGCT 1320 CTGCTGAAGC CAGTTACCTT CGGAAAAAGA GTTGGTAGCTCTTGATCCGG CAAACAAACC 1380 ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC AAGCAGCAGATTACGCGCAG AAAAAAAGGA 1440 TCTCAAGAAG ATCCTTTGAT CTTTTCTACG GGGTCTGACGCTCAGTGGAA CGAAAACTCA 1500 CGTTAAGGGA TTTTGGTCAT GAGATTATCG TCGACAATTCGCGCGCGAAG GCGAAGCGGC 1560 ATGCATTTAC GTTGACACCA TCGAATGGTG CAAAACCTTTCGCGGTATGG CATGATAGCG 1620 CCCGGAAGAG AGTCAATTCA GGGTGGTGAA TGTGAAACCAGTAACGTTAT ACGATGTCGC 1680 AGAGTATGCC GGTGTCTCTT ATCAGACCGT TTCCCGCGTGGTGAACCAGG CCAGCCACGT 1740 TTCTGCGAAA ACGCGGGAAA AAGTGGAAGC GGCGATGGCGGAGCTGAATT ACATTCCCAA 1800 CCGCGTGGCA CAACAACTGG CGGGCAAACA GTCGTTGCTGATTGGCGTTG CCACCTCCAG 1860 TCTGGCCCTG CACGCGCCGT CGCAAATTGT CGCGGCGATTAAATCTCGCG CCGATCAACT 1920 GGGTGCCAGC GTGGTGGTGT CGATGGTAGA ACGAAGCGGCGTCGAAGCCT GTAAAGCGGC 1980 GGTGCACAAT CTTCTCGCGC AACGCGTCAG TGGGCTGATCATTAACTATC CGCTGGATGA 2040 CCAGGATGCC ATTGCTGTGG AAGCTGCCTG CACTAATGTTCCGGCGTTAT TTCTTGATGT 2100 CTCTGACCAG ACACCCATCA ACAGTATTAT TTTCTCCCATGAAGACGGTA CGCGACTGGG 2160 CGTGGAGCAT CTGGTCGCAT TGGGTCACCA GCAAATCGCGCTGTTAGCGG GCCCATTAAG 2220 TTCTGTCTCG GCGCGTCTGC GTCTGGCTGG CTGGCATAAATATCTCACTC GCAATCAAAT 2280 TCAGCCGATA GCGGAACGGG AAGGCGACTG GAGTGCCATGTCCGGTTTTC AACAAACCAT 2340 GCAAATGCTG AATGAGGGCA TCGTTCCCAC TGCGATGCTGGTTGCCAACG ATCAGATGGC 2400 GCTGGGCGCA ATGCGCGCCA TTACCGAGTC CGGGCTGCGCGTTGGTGCGG ATATCTCGGT 2460 AGTGGGATAC GACGATACCG AAGACAGCTC ATGTTATATCCCGCCGTTAA CCACCATCAA 2520 ACAGGATTTT CGCCTGCTGG GGCAAACCAG CGTGGACCGCTTGCTGCAAC TCTCTCAGGG 2580 CCAGGCGGTG AAGGGCAATC AGCTGTTGCC CGTCTCACTGGTGAAAAGAA AAACCACCCT 2640 GGCGCCCAAT ACGCAAACCG CCTCTCCCCG CGCGTTGGCCGATTCATTAA TGCAGCTGGC 2700 ACGACAGGTT TCCCGACTGG AAAGCGGGCA GTGAGCGCAACGCAATTAAT GTAAGTTAGC 2760 GCGAATTGTC GACCAAAGCG GCCATCGTGC CTCCCCACTCCTGCAGTTCG GGGGCATGGA 2820 TGCGCGGATA GCCGCTGCTG GTTTCCTGGA TGCCGACGGATTTGCACTGC CGGTAGAACT 2880 CCGCGAGGTC GTCCAGCCTC AGGCAGCAGC TGAACCAACTCGCGAGGGGA TCGAGCCCGG 2940 GGTGGGCGAA GAACTCCAGC ATGAGATCCC CGCGCTGGAGGATCATCCAG CCGGCGTCCC 3000 GGAAAACGAT TCCGAAGCCC AACCTTTCAT AGAAGGCGGCGGTGGAATCG AAATCTCGTG 3060 ATGGCAGGTT GGGCGTCGCT TGGTCGGTCA TTTCGAACCCCAGAGTCCCG CTCAGAAGAA 3120 CTCGTCAAGA AGGCGATAGA AGGCGATGCG CTGCGAATCGGGAGCGGCGA TACCGTAAAG 3180 CACGAGGAAG CGGTCAGCCC ATTCGCCGCC AAGCTCTTCAGCAATATCAC GGGTAGCCAA 3240 CGCTATGTCC TGATAGCGGT CCGCCACACC CAGCCGGCCACAGTCGATGA ATCCAGAAAA 3300 GCGGCCATTT TCCACCATGA TATTCGGCAA GCAGGCATCGCCATGGGTCA CGACGAGATC 3360 CTCGCCGTCG GGCATGCGCG CCTTGAGCCT GGCGAACAGTTCGGCTGGCG CGAGCCCCTG 3420 ATGCTCTTCG TCCAGATCAT CCTGATCGAC AAGACCGGCTTCCATCCGAG TACGTGCTCG 3480 CTCGATGCGA TGTTTCGCTT GGTGGTCGAA TGGGCAGGTAGCCGGATCAA GCGTATGCAG 3540 CCGCCGCATT GCATCAGCCA TGATGGATAC TTTCTCGGCAGGAGCAAGGT GAGATGACAG 3600 GAGATCCTGC CCCGGCACTT CGCCCAATAG CAGCCAGTCCCTTCCCGCTT CAGTGACAAC 3660 GTCGAGCACA GCTGCGCAAG GAACGCCCGT CGTGGCCAGCCACGATAGCC GCGCTGCCTC 3720 GTCCTGCAGT TCATTCAGGG CACCGGACAG GTCGGTCTTGACAAAAAGAA CCGGGCGCCC 3780 CTGCGCTGAC AGCCGGAACA CGGCGGCATC AGAGCAGCCGATTGTCTGTT GTGCCCAGTC 3840 ATAGCCGAAT AGCCTCTCCA CCCAAGCGGC CGGAGAACCTGCGTGCAATC CATCTTGTTC 3900 AATCATGCGA AACGATCCTC ATCCTGTCTC TTGATCAGATCTTGATCCCC TGCGCCATCA 3960 GATCCTTGGC GGCAAGAAAG CCATCCAGTT TACTTTGCAGGGCTTCCCAA CCTTACCAGA 4020 GGGCGCCCCA GCTGGCAATT CCGGTTCGCT TGCTGTCCATAAAACCGCCC AGTCTAGCTA 4080 TCGCCATGTA AGCCCACTGC AAGCTACCTG CTTTCTCTTTGCGCTTGCGT TTTCCCTTGT 4140 CCAGATAGCC CAGTAGCTGA CATTCATCCG GGGTCAGCACCGTTTCTGCG GACTGGCTTT 4200 CTACGTGTTC CGCTTCCTTT AGCAGCCCTT GCGCCCTGAGTGCTTGCGGC AGCGTG 4256 112 base pairs nucleic acid double linear cDNA 22AAGCTTAAAA AACTGCAAAA AATAGTTTGA CTTGTGAGCG GATAACAATT AAGATGTACC 60CAATTGTGAG CGGATAACAA TTTCACACAT TAAAGAGGAG AAATTACATA TG 112 45 aminoacids amino acid linear protein 23 Pro Ser Pro Cys Cys Met Phe Phe ValSer Lys Arg Ile Pro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu SerSer Arg Ser Thr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys LysGly Gln Gln Phe Cys 35 40 45 46 amino acids amino acid linear protein 24Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu Asn 1 5 1015 Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys Ala 20 2530 Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly 35 40 45 47amino acids amino acid linear protein 25 Pro Ser Pro Cys Cys Met Phe PheVal Ser Lys Arg Ile Pro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln LeuSer Ser Arg Ser Thr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr LysLys Gly Gln Gln Phe Cys Gly Asp 35 40 45 48 amino acids amino acidlinear protein 26 Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg IlePro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser ThrCys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln PheCys Gly Asp Pro 35 40 45 49 amino acids amino acid linear protein 27 ProSer Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu Asn 1 5 10 15Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys Ala 20 25 30Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp Pro 35 40 45Lys 50 amino acids amino acid linear protein 28 Pro Ser Pro Cys Cys MetPhe Phe Val Ser Lys Arg Ile Pro Glu Asn 1 5 10 15 Arg Val Val Ser TyrGln Leu Ser Ser Arg Ser Thr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe ThrThr Lys Lys Gly Gln Gln Phe Cys Gly Asp Pro 35 40 45 Lys Gln 50 51 aminoacids amino acid linear protein 29 Pro Ser Pro Cys Cys Met Phe Phe ValSer Lys Arg Ile Pro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu SerSer Arg Ser Thr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys LysGly Gln Gln Phe Cys Gly Asp Pro 35 40 45 Lys Gln Glu 50 52 amino acidsamino acid linear protein 30 Pro Ser Pro Cys Cys Met Phe Phe Val Ser LysArg Ile Pro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu Ser Ser ArgSer Thr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys Lys Gly GlnGln Phe Cys Gly Asp Pro 35 40 45 Lys Gln Glu Trp 50 53 amino acids aminoacid linear protein 31 Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys ArgIle Pro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu Ser Ser Arg SerThr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys Lys Gly Gln GlnPhe Cys Gly Asp Pro 35 40 45 Lys Gln Glu Trp Val 50 54 amino acids aminoacid linear protein 32 Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys ArgIle Pro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu Ser Ser Arg SerThr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys Lys Gly Gln GlnPhe Cys Gly Asp Pro 35 40 45 Lys Gln Glu Trp Val Gln 50 55 amino acidsamino acid linear protein 33 Pro Ser Pro Cys Cys Met Phe Phe Val Ser LysArg Ile Pro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu Ser Ser ArgSer Thr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys Lys Gly GlnGln Phe Cys Gly Asp Pro 35 40 45 Lys Gln Glu Trp Val Gln Arg 50 55 56amino acids amino acid linear protein 34 Pro Ser Pro Cys Cys Met Phe PheVal Ser Lys Arg Ile Pro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln LeuSer Ser Arg Ser Thr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr LysLys Gly Gln Gln Phe Cys Gly Asp Pro 35 40 45 Lys Gln Glu Trp Val Gln ArgTyr 50 55 57 amino acids amino acid linear protein 35 Pro Ser Pro CysCys Met Phe Phe Val Ser Lys Arg Ile Pro Glu Asn 1 5 10 15 Arg Val ValSer Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys Ala 20 25 30 Gly Val IlePhe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp Pro 35 40 45 Lys Gln GluTrp Val Gln Arg Tyr Met 50 55 58 amino acids amino acid linear protein36 Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu Asn 1 510 15 Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys Ala 2025 30 Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp Pro 3540 45 Lys Gln Glu Trp Val Gln Arg Tyr Met Lys 50 55 59 amino acids aminoacid linear protein 37 Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys ArgIle Pro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu Ser Ser Arg SerThr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys Lys Gly Gln GlnPhe Cys Gly Asp Pro 35 40 45 Lys Gln Glu Trp Val Gln Arg Tyr Met Lys Asn50 55 60 amino acids amino acid linear protein 38 Pro Ser Pro Cys CysMet Phe Phe Val Ser Lys Arg Ile Pro Glu Asn 1 5 10 15 Arg Val Val SerTyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys Ala 20 25 30 Gly Val Ile PheThr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp Pro 35 40 45 Lys Gln Glu TrpVal Gln Arg Tyr Met Lys Asn Leu 50 55 60 61 amino acids amino acidlinear protein 39 Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg IlePro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser ThrCys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln PheCys Gly Asp Pro 35 40 45 Lys Gln Glu Trp Val Gln Arg Tyr Met Lys Asn LeuAsp 50 55 60 62 amino acids amino acid linear protein 40 Pro Ser Pro CysCys Met Phe Phe Val Ser Lys Arg Ile Pro Glu Asn 1 5 10 15 Arg Val ValSer Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys Ala 20 25 30 Gly Val IlePhe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp Pro 35 40 45 Lys Gln GluTrp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala 50 55 60 63 amino acidsamino acid linear protein 41 Pro Ser Pro Cys Cys Met Phe Phe Val Ser LysArg Ile Pro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu Ser Ser ArgSer Thr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys Lys Gly GlnGln Phe Cys Gly Asp Pro 35 40 45 Lys Gln Glu Trp Val Gln Arg Tyr Met LysAsn Leu Asp Ala Lys 50 55 60 64 amino acids amino acid linear protein 42Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu Asn 1 5 1015 Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys Ala 20 2530 Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp Pro 35 4045 Lys Gln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys Gln 50 5560 65 amino acids amino acid linear protein 43 Pro Ser Pro Cys Cys MetPhe Phe Val Ser Lys Arg Ile Pro Glu Asn 1 5 10 15 Arg Val Val Ser TyrGln Leu Ser Ser Arg Ser Thr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe ThrThr Lys Lys Gly Gln Gln Phe Cys Gly Asp Pro 35 40 45 Lys Gln Glu Trp ValGln Arg Tyr Met Lys Asn Leu Asp Ala Lys Gln 50 55 60 Lys 65 66 aminoacids amino acid linear protein 44 Pro Ser Pro Cys Cys Met Phe Phe ValSer Lys Arg Ile Pro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu SerSer Arg Ser Thr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys LysGly Gln Gln Phe Cys Gly Asp Pro 35 40 45 Lys Gln Glu Trp Val Gln Arg TyrMet Lys Asn Leu Asp Ala Lys Gln 50 55 60 Lys Lys 65 67 amino acids aminoacid linear protein 45 Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys ArgIle Pro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu Ser Ser Arg SerThr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys Lys Gly Gln GlnPhe Cys Gly Asp Pro 35 40 45 Lys Gln Glu Trp Val Gln Arg Tyr Met Lys AsnLeu Asp Ala Lys Gln 50 55 60 Lys Lys Ala 65 68 amino acids amino acidlinear protein 46 Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg IlePro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser ThrCys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln PheCys Gly Asp Pro 35 40 45 Lys Gln Glu Trp Val Gln Arg Tyr Met Lys Asn LeuAsp Ala Lys Gln 50 55 60 Lys Lys Ala Ser 65 69 amino acids amino acidlinear protein 47 Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg IlePro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser ThrCys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln PheCys Gly Asp Pro 35 40 45 Lys Gln Glu Trp Val Gln Arg Tyr Met Lys Asn LeuAsp Ala Lys Gln 50 55 60 Lys Lys Ala Ser Pro 65 70 amino acids aminoacid linear protein 48 Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys ArgIle Pro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu Ser Ser Arg SerThr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys Lys Gly Gln GlnPhe Cys Gly Asp Pro 35 40 45 Lys Gln Glu Trp Val Gln Arg Tyr Met Lys AsnLeu Asp Ala Lys Gln 50 55 60 Lys Lys Ala Ser Pro Arg 65 70 71 aminoacids amino acid linear protein 49 Pro Ser Pro Cys Cys Met Phe Phe ValSer Lys Arg Ile Pro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu SerSer Arg Ser Thr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys LysGly Gln Gln Phe Cys Gly Asp Pro 35 40 45 Lys Gln Glu Trp Val Gln Arg TyrMet Lys Asn Leu Asp Ala Lys Gln 50 55 60 Lys Lys Ala Ser Pro Arg Ala 6570 72 amino acids amino acid linear protein 50 Pro Ser Pro Cys Cys MetPhe Phe Val Ser Lys Arg Ile Pro Glu Asn 1 5 10 15 Arg Val Val Ser TyrGln Leu Ser Ser Arg Ser Thr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe ThrThr Lys Lys Gly Gln Gln Phe Cys Gly Asp Pro 35 40 45 Lys Gln Glu Trp ValGln Arg Tyr Met Lys Asn Leu Asp Ala Lys Gln 50 55 60 Lys Lys Ala Ser ProArg Ala Arg 65 70 73 amino acids amino acid linear protein 51 Pro SerPro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu Asn 1 5 10 15 ArgVal Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys Ala 20 25 30 GlyVal Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp Pro 35 40 45 LysGln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys Gln 50 55 60 LysLys Ala Ser Pro Arg Ala Arg Ala 65 70 74 amino acids amino acid linearprotein 52 Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro GluAsn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys LeuLys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys GlyAsp Pro 35 40 45 Lys Gln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu Asp AlaLys Gln 50 55 60 Lys Lys Ala Ser Pro Arg Ala Arg Ala Val 65 70 75 aminoacids amino acid linear protein 53 Pro Ser Pro Cys Cys Met Phe Phe ValSer Lys Arg Ile Pro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu SerSer Arg Ser Thr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys LysGly Gln Gln Phe Cys Gly Asp Pro 35 40 45 Lys Gln Glu Trp Val Gln Arg TyrMet Lys Asn Leu Asp Ala Lys Gln 50 55 60 Lys Lys Ala Ser Pro Arg Ala ArgAla Val Ala 65 70 75 76 amino acids amino acid linear protein 54 Pro SerPro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu Asn 1 5 10 15 ArgVal Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys Ala 20 25 30 GlyVal Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp Pro 35 40 45 LysGln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys Gln 50 55 60 LysLys Ala Ser Pro Arg Ala Arg Ala Val Ala Val 65 70 75 77 amino acidsamino acid linear protein 55 Pro Ser Pro Cys Cys Met Phe Phe Val Ser LysArg Ile Pro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu Ser Ser ArgSer Thr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys Lys Gly GlnGln Phe Cys Gly Asp Pro 35 40 45 Lys Gln Glu Trp Val Gln Arg Tyr Met LysAsn Leu Asp Ala Lys Gln 50 55 60 Lys Lys Ala Ser Pro Arg Ala Arg Ala ValAla Val Lys 65 70 75 78 amino acids amino acid linear protein 56 Pro SerPro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu Asn 1 5 10 15 ArgVal Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys Ala 20 25 30 GlyVal Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp Pro 35 40 45 LysGln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys Gln 50 55 60 LysLys Ala Ser Pro Arg Ala Arg Ala Val Ala Val Lys Gly 65 70 75 79 aminoacids amino acid linear protein 57 Pro Ser Pro Cys Cys Met Phe Phe ValSer Lys Arg Ile Pro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu SerSer Arg Ser Thr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys LysGly Gln Gln Phe Cys Gly Asp Pro 35 40 45 Lys Gln Glu Trp Val Gln Arg TyrMet Lys Asn Leu Asp Ala Lys Gln 50 55 60 Lys Lys Ala Ser Pro Arg Ala ArgAla Val Ala Val Lys Gly Pro 65 70 75 80 amino acids amino acid linearprotein 58 Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro GluAsn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys LeuLys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys GlyAsp Pro 35 40 45 Lys Gln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu Asp AlaLys Gln 50 55 60 Lys Lys Ala Ser Pro Arg Ala Arg Ala Val Ala Val Lys GlyPro Val 65 70 75 80 81 amino acids amino acid linear protein 59 Pro SerPro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu Asn 1 5 10 15 ArgVal Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys Ala 20 25 30 GlyVal Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp Pro 35 40 45 LysGln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys Gln 50 55 60 LysLys Ala Ser Pro Arg Ala Arg Ala Val Ala Val Lys Gly Pro Val 65 70 75 80Gln 82 amino acids amino acid linear protein 60 Pro Ser Pro Cys Cys MetPhe Phe Val Ser Lys Arg Ile Pro Glu Asn 1 5 10 15 Arg Val Val Ser TyrGln Leu Ser Ser Arg Ser Thr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe ThrThr Lys Lys Gly Gln Gln Phe Cys Gly Asp Pro 35 40 45 Lys Gln Glu Trp ValGln Arg Tyr Met Lys Asn Leu Asp Ala Lys Gln 50 55 60 Lys Lys Ala Ser ProArg Ala Arg Ala Val Ala Val Lys Gly Pro Val 65 70 75 80 Gln Arg 83 aminoacids amino acid linear protein 61 Pro Ser Pro Cys Cys Met Phe Phe ValSer Lys Arg Ile Pro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu SerSer Arg Ser Thr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys LysGly Gln Gln Phe Cys Gly Asp Pro 35 40 45 Lys Gln Glu Trp Val Gln Arg TyrMet Lys Asn Leu Asp Ala Lys Gln 50 55 60 Lys Lys Ala Ser Pro Arg Ala ArgAla Val Ala Val Lys Gly Pro Val 65 70 75 80 Gln Arg Tyr 84 amino acidsamino acid linear protein 62 Pro Ser Pro Cys Cys Met Phe Phe Val Ser LysArg Ile Pro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu Ser Ser ArgSer Thr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys Lys Gly GlnGln Phe Cys Gly Asp Pro 35 40 45 Lys Gln Glu Trp Val Gln Arg Tyr Met LysAsn Leu Asp Ala Lys Gln 50 55 60 Lys Lys Ala Ser Pro Arg Ala Arg Ala ValAla Val Lys Gly Pro Val 65 70 75 80 Gln Arg Tyr Pro 85 amino acids aminoacid linear protein 63 Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys ArgIle Pro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu Ser Ser Arg SerThr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys Lys Gly Gln GlnPhe Cys Gly Asp Pro 35 40 45 Lys Gln Glu Trp Val Gln Arg Tyr Met Lys AsnLeu Asp Ala Lys Gln 50 55 60 Lys Lys Ala Ser Pro Arg Ala Arg Ala Val AlaVal Lys Gly Pro Val 65 70 75 80 Gln Arg Tyr Pro Gly 85 86 amino acidsamino acid linear protein 64 Pro Ser Pro Cys Cys Met Phe Phe Val Ser LysArg Ile Pro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu Ser Ser ArgSer Thr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys Lys Gly GlnGln Phe Cys Gly Asp Pro 35 40 45 Lys Gln Glu Trp Val Gln Arg Tyr Met LysAsn Leu Asp Ala Lys Gln 50 55 60 Lys Lys Ala Ser Pro Arg Ala Arg Ala ValAla Val Lys Gly Pro Val 65 70 75 80 Gln Arg Tyr Pro Gly Asn 85 87 aminoacids amino acid linear protein 65 Pro Ser Pro Cys Cys Met Phe Phe ValSer Lys Arg Ile Pro Glu Asn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu SerSer Arg Ser Thr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys LysGly Gln Gln Phe Cys Gly Asp Pro 35 40 45 Lys Gln Glu Trp Val Gln Arg TyrMet Lys Asn Leu Asp Ala Lys Gln 50 55 60 Lys Lys Ala Ser Pro Arg Ala ArgAla Val Ala Val Lys Gly Pro Val 65 70 75 80 Gln Arg Tyr Pro Gly Asn Gln85 88 amino acids amino acid linear protein 66 Pro Ser Pro Cys Cys MetPhe Phe Val Ser Lys Arg Ile Pro Glu Asn 1 5 10 15 Arg Val Val Ser TyrGln Leu Ser Ser Arg Ser Thr Cys Leu Lys Ala 20 25 30 Gly Val Ile Phe ThrThr Lys Lys Gly Gln Gln Phe Cys Gly Asp Pro 35 40 45 Lys Gln Glu Trp ValGln Arg Tyr Met Lys Asn Leu Asp Ala Lys Gln 50 55 60 Lys Lys Ala Ser ProArg Ala Arg Ala Val Ala Val Lys Gly Pro Val 65 70 75 80 Gln Arg Tyr ProGly Asn Gln Thr 85 89 amino acids amino acid linear protein 67 Pro SerPro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu Asn 1 5 10 15 ArgVal Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys Ala 20 25 30 GlyVal Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp Pro 35 40 45 LysGln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys Gln 50 55 60 LysLys Ala Ser Pro Arg Ala Arg Ala Val Ala Val Lys Gly Pro Val 65 70 75 80Gln Arg Tyr Pro Gly Asn Gln Thr Thr 85 90 amino acids amino acid linearprotein 68 Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro GluAsn 1 5 10 15 Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys LeuLys Ala 20 25 30 Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys GlyAsp Pro 35 40 45 Lys Gln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu Asp AlaLys Gln 50 55 60 Lys Lys Ala Ser Pro Arg Ala Arg Ala Val Ala Val Lys GlyPro Val 65 70 75 80 Gln Arg Tyr Pro Gly Asn Gln Thr Thr Cys 85 90 46amino acids amino acid linear protein 69 Met Pro Ser Pro Cys Cys Met PhePhe Val Ser Lys Arg Ile Pro Gl 1 5 10 15 Asn Arg Val Val Ser Tyr Gln LeuSer Ser Arg Ser Thr Cys Leu Ly 20 25 30 Ala Gly Val Ile Phe Thr Thr LysLys Gly Gln Gln Phe Cys 35 40 45 47 amino acids amino acid linearprotein 70 Met Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile ProGlu 1 5 10 15 Asn Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr CysLeu Lys 20 25 30 Ala Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe CysGly 35 40 45 48 amino acids amino acid linear protein 71 Met Pro Ser ProCys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu 1 5 10 15 Asn Arg ValVal Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 20 25 30 Ala Gly ValIle Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 35 40 45 49 aminoacids amino acid linear protein 72 Met Pro Ser Pro Cys Cys Met Phe PheVal Ser Lys Arg Ile Pro Glu 1 5 10 15 Asn Arg Val Val Ser Tyr Gln LeuSer Ser Arg Ser Thr Cys Leu Lys 20 25 30 Ala Gly Val Ile Phe Thr Thr LysLys Gly Gln Gln Phe Cys Gly Asp 35 40 45 Pro 50 amino acids amino acidlinear protein 73 Met Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys ArgIle Pro Glu 1 5 10 15 Asn Arg Val Val Ser Tyr Gln Leu Ser Ser Arg SerThr Cys Leu Lys 20 25 30 Ala Gly Val Ile Phe Thr Thr Lys Lys Gly Gln GlnPhe Cys Gly Asp 35 40 45 Pro Lys 50 51 amino acids amino acid linearprotein 74 Met Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile ProGlu 1 5 10 15 Asn Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr CysLeu Lys 20 25 30 Ala Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe CysGly Asp 35 40 45 Pro Lys Gln 50 52 amino acids amino acid linear protein75 Met Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu 1 510 15 Asn Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 2025 30 Ala Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 3540 45 Pro Lys Gln Glu 50 53 amino acids amino acid linear protein 76 MetPro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu 1 5 10 15Asn Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 20 25 30Ala Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 35 40 45Pro Lys Gln Glu Trp 50 54 amino acids amino acid linear protein 77 MetPro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu 1 5 10 15Asn Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 20 25 30Ala Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 35 40 45Pro Lys Gln Glu Trp Val 50 55 amino acids amino acid linear protein 78Met Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu 1 5 1015 Asn Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 20 2530 Ala Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 35 4045 Pro Lys Gln Glu Trp Val Gln 50 55 56 amino acids amino acid linearprotein 79 Met Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile ProGlu 1 5 10 15 Asn Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr CysLeu Lys 20 25 30 Ala Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe CysGly Asp 35 40 45 Pro Lys Gln Glu Trp Val Gln Arg 50 55 57 amino acidsamino acid linear protein 80 Met Pro Ser Pro Cys Cys Met Phe Phe Val SerLys Arg Ile Pro Glu 1 5 10 15 Asn Arg Val Val Ser Tyr Gln Leu Ser SerArg Ser Thr Cys Leu Lys 20 25 30 Ala Gly Val Ile Phe Thr Thr Lys Lys GlyGln Gln Phe Cys Gly Asp 35 40 45 Pro Lys Gln Glu Trp Val Gln Arg Tyr 5055 58 amino acids amino acid linear protein 81 Met Pro Ser Pro Cys CysMet Phe Phe Val Ser Lys Arg Ile Pro Glu 1 5 10 15 Asn Arg Val Val SerTyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 20 25 30 Ala Gly Val Ile PheThr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 35 40 45 Pro Lys Gln Glu TrpVal Gln Arg Tyr Met 50 55 59 amino acids amino acid linear protein 82Met Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu 1 5 1015 Asn Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 20 2530 Ala Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 35 4045 Pro Lys Gln Glu Trp Val Gln Arg Tyr Met Lys 50 55 60 amino acidsamino acid linear protein 83 Met Pro Ser Pro Cys Cys Met Phe Phe Val SerLys Arg Ile Pro Glu 1 5 10 15 Asn Arg Val Val Ser Tyr Gln Leu Ser SerArg Ser Thr Cys Leu Lys 20 25 30 Ala Gly Val Ile Phe Thr Thr Lys Lys GlyGln Gln Phe Cys Gly Asp 35 40 45 Pro Lys Gln Glu Trp Val Gln Arg Tyr MetLys Asn 50 55 60 61 amino acids amino acid linear protein 84 Met Pro SerPro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu 1 5 10 15 Asn ArgVal Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 20 25 30 Ala GlyVal Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 35 40 45 Pro LysGln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu 50 55 60 62 amino acidsamino acid linear protein 85 Met Pro Ser Pro Cys Cys Met Phe Phe Val SerLys Arg Ile Pro Glu 1 5 10 15 Asn Arg Val Val Ser Tyr Gln Leu Ser SerArg Ser Thr Cys Leu Lys 20 25 30 Ala Gly Val Ile Phe Thr Thr Lys Lys GlyGln Gln Phe Cys Gly Asp 35 40 45 Pro Lys Gln Glu Trp Val Gln Arg Tyr MetLys Asn Leu Asp 50 55 60 63 amino acids amino acid linear protein 86 MetPro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu 1 5 10 15Asn Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 20 25 30Ala Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 35 40 45Pro Lys Gln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala 50 55 60 64amino acids amino acid linear protein 87 Met Pro Ser Pro Cys Cys Met PhePhe Val Ser Lys Arg Ile Pro Glu 1 5 10 15 Asn Arg Val Val Ser Tyr GlnLeu Ser Ser Arg Ser Thr Cys Leu Lys 20 25 30 Ala Gly Val Ile Phe Thr ThrLys Lys Gly Gln Gln Phe Cys Gly Asp 35 40 45 Pro Lys Gln Glu Trp Val GlnArg Tyr Met Lys Asn Leu Asp Ala Lys 50 55 60 65 amino acids amino acidlinear protein 88 Met Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys ArgIle Pro Glu 1 5 10 15 Asn Arg Val Val Ser Tyr Gln Leu Ser Ser Arg SerThr Cys Leu Lys 20 25 30 Ala Gly Val Ile Phe Thr Thr Lys Lys Gly Gln GlnPhe Cys Gly Asp 35 40 45 Pro Lys Gln Glu Trp Val Gln Arg Tyr Met Lys AsnLeu Asp Ala Lys 50 55 60 Gln 65 66 amino acids amino acid linear protein89 Met Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu 1 510 15 Asn Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 2025 30 Ala Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 3540 45 Pro Lys Gln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys 5055 60 Gln Lys 65 67 amino acids amino acid linear protein 90 Met Pro SerPro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu 1 5 10 15 Asn ArgVal Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 20 25 30 Ala GlyVal Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 35 40 45 Pro LysGln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys 50 55 60 Gln LysLys 65 68 amino acids amino acid linear protein 91 Met Pro Ser Pro CysCys Met Phe Phe Val Ser Lys Arg Ile Pro Glu 1 5 10 15 Asn Arg Val ValSer Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 20 25 30 Ala Gly Val IlePhe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 35 40 45 Pro Lys Gln GluTrp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys 50 55 60 Gln Lys Lys Ala65 69 amino acids amino acid linear protein 92 Met Pro Ser Pro Cys CysMet Phe Phe Val Ser Lys Arg Ile Pro Glu 1 5 10 15 Asn Arg Val Val SerTyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 20 25 30 Ala Gly Val Ile PheThr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 35 40 45 Pro Lys Gln Glu TrpVal Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys 50 55 60 Gln Lys Lys Ala Ser65 70 amino acids amino acid linear protein 93 Met Pro Ser Pro Cys CysMet Phe Phe Val Ser Lys Arg Ile Pro Glu 1 5 10 15 Asn Arg Val Val SerTyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 20 25 30 Ala Gly Val Ile PheThr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 35 40 45 Pro Lys Gln Glu TrpVal Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys 50 55 60 Gln Lys Lys Ala SerPro 65 70 71 amino acids amino acid linear protein 94 Met Pro Ser ProCys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu 1 5 10 15 Asn Arg ValVal Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 20 25 30 Ala Gly ValIle Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 35 40 45 Pro Lys GlnGlu Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys 50 55 60 Gln Lys LysAla Ser Pro Arg 65 70 72 amino acids amino acid linear protein 95 MetPro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu 1 5 10 15Asn Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 20 25 30Ala Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 35 40 45Pro Lys Gln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys 50 55 60Gln Lys Lys Ala Ser Pro Arg Ala 65 70 73 amino acids amino acid linearprotein 96 Met Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile ProGlu 1 5 10 15 Asn Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr CysLeu Lys 20 25 30 Ala Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe CysGly Asp 35 40 45 Pro Lys Gln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu AspAla Lys 50 55 60 Gln Lys Lys Ala Ser Pro Arg Ala Arg 65 70 74 aminoacids amino acid linear protein 97 Met Pro Ser Pro Cys Cys Met Phe PheVal Ser Lys Arg Ile Pro Glu 1 5 10 15 Asn Arg Val Val Ser Tyr Gln LeuSer Ser Arg Ser Thr Cys Leu Lys 20 25 30 Ala Gly Val Ile Phe Thr Thr LysLys Gly Gln Gln Phe Cys Gly Asp 35 40 45 Pro Lys Gln Glu Trp Val Gln ArgTyr Met Lys Asn Leu Asp Ala Lys 50 55 60 Gln Lys Lys Ala Ser Pro Arg AlaArg Ala 65 70 75 amino acids amino acid linear protein 98 Met Pro SerPro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu 1 5 10 15 Asn ArgVal Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 20 25 30 Ala GlyVal Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 35 40 45 Pro LysGln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys 50 55 60 Gln LysLys Ala Ser Pro Arg Ala Arg Ala Val 65 70 75 76 amino acids amino acidlinear protein 99 Met Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys ArgIle Pro Glu 1 5 10 15 Asn Arg Val Val Ser Tyr Gln Leu Ser Ser Arg SerThr Cys Leu Lys 20 25 30 Ala Gly Val Ile Phe Thr Thr Lys Lys Gly Gln GlnPhe Cys Gly Asp 35 40 45 Pro Lys Gln Glu Trp Val Gln Arg Tyr Met Lys AsnLeu Asp Ala Lys 50 55 60 Gln Lys Lys Ala Ser Pro Arg Ala Arg Ala Val Ala65 70 75 77 amino acids amino acid linear protein 100 Met Pro Ser ProCys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu 1 5 10 15 Asn Arg ValVal Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 20 25 30 Ala Gly ValIle Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 35 40 45 Pro Lys GlnGlu Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys 50 55 60 Gln Lys LysAla Ser Pro Arg Ala Arg Ala Val Ala Val 65 70 75 78 amino acids aminoacid linear protein 101 Met Pro Ser Pro Cys Cys Met Phe Phe Val Ser LysArg Ile Pro Glu 1 5 10 15 Asn Arg Val Val Ser Tyr Gln Leu Ser Ser ArgSer Thr Cys Leu Lys 20 25 30 Ala Gly Val Ile Phe Thr Thr Lys Lys Gly GlnGln Phe Cys Gly Asp 35 40 45 Pro Lys Gln Glu Trp Val Gln Arg Tyr Met LysAsn Leu Asp Ala Lys 50 55 60 Gln Lys Lys Ala Ser Pro Arg Ala Arg Ala ValAla Val Lys 65 70 75 79 amino acids amino acid linear protein 102 MetPro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu 1 5 10 15Asn Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 20 25 30Ala Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 35 40 45Pro Lys Gln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys 50 55 60Gln Lys Lys Ala Ser Pro Arg Ala Arg Ala Val Ala Val Lys Gly 65 70 75 80amino acids amino acid linear protein 103 Met Pro Ser Pro Cys Cys MetPhe Phe Val Ser Lys Arg Ile Pro Glu 1 5 10 15 Asn Arg Val Val Ser TyrGln Leu Ser Ser Arg Ser Thr Cys Leu Lys 20 25 30 Ala Gly Val Ile Phe ThrThr Lys Lys Gly Gln Gln Phe Cys Gly Asp 35 40 45 Pro Lys Gln Glu Trp ValGln Arg Tyr Met Lys Asn Leu Asp Ala Lys 50 55 60 Gln Lys Lys Ala Ser ProArg Ala Arg Ala Val Ala Val Lys Gly Pro 65 70 75 80 81 amino acids aminoacid linear protein 104 Met Pro Ser Pro Cys Cys Met Phe Phe Val Ser LysArg Ile Pro Glu 1 5 10 15 Asn Arg Val Val Ser Tyr Gln Leu Ser Ser ArgSer Thr Cys Leu Lys 20 25 30 Ala Gly Val Ile Phe Thr Thr Lys Lys Gly GlnGln Phe Cys Gly Asp 35 40 45 Pro Lys Gln Glu Trp Val Gln Arg Tyr Met LysAsn Leu Asp Ala Lys 50 55 60 Gln Lys Lys Ala Ser Pro Arg Ala Arg Ala ValAla Val Lys Gly Pro 65 70 75 80 Val 82 amino acids amino acid linearprotein 105 Met Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile ProGlu 1 5 10 15 Asn Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr CysLeu Lys 20 25 30 Ala Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe CysGly Asp 35 40 45 Pro Lys Gln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu AspAla Lys 50 55 60 Gln Lys Lys Ala Ser Pro Arg Ala Arg Ala Val Ala Val LysGly Pro 65 70 75 80 Val Gln 83 amino acids amino acid linear protein 106Met Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu 1 5 1015 Asn Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 20 2530 Ala Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 35 4045 Pro Lys Gln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys 50 5560 Gln Lys Lys Ala Ser Pro Arg Ala Arg Ala Val Ala Val Lys Gly Pro 65 7075 80 Val Gln Arg 84 amino acids amino acid linear protein 107 Met ProSer Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu 1 5 10 15 AsnArg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 20 25 30 AlaGly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 35 40 45 ProLys Gln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys 50 55 60 GlnLys Lys Ala Ser Pro Arg Ala Arg Ala Val Ala Val Lys Gly Pro 65 70 75 80Val Gln Arg Tyr 85 amino acids amino acid linear protein 108 Met Pro SerPro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu 1 5 10 15 Asn ArgVal Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 20 25 30 Ala GlyVal Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 35 40 45 Pro LysGln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys 50 55 60 Gln LysLys Ala Ser Pro Arg Ala Arg Ala Val Ala Val Lys Gly Pro 65 70 75 80 ValGln Arg Tyr Pro 85 86 amino acids amino acid linear protein 109 Met ProSer Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu 1 5 10 15 AsnArg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 20 25 30 AlaGly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 35 40 45 ProLys Gln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys 50 55 60 GlnLys Lys Ala Ser Pro Arg Ala Arg Ala Val Ala Val Lys Gly Pro 65 70 75 80Val Gln Arg Tyr Pro Gly 85 87 amino acids amino acid linear protein 110Met Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu 1 5 1015 Asn Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 20 2530 Ala Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 35 4045 Pro Lys Gln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys 50 5560 Gln Lys Lys Ala Ser Pro Arg Ala Arg Ala Val Ala Val Lys Gly Pro 65 7075 80 Val Gln Arg Tyr Pro Gly Asn 85 88 amino acids amino acid linearprotein 111 Met Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile ProGlu 1 5 10 15 Asn Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr CysLeu Lys 20 25 30 Ala Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe CysGly Asp 35 40 45 Pro Lys Gln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu AspAla Lys 50 55 60 Gln Lys Lys Ala Ser Pro Arg Ala Arg Ala Val Ala Val LysGly Pro 65 70 75 80 Val Gln Arg Tyr Pro Gly Asn Gln 85 89 amino acidsamino acid linear protein 112 Met Pro Ser Pro Cys Cys Met Phe Phe ValSer Lys Arg Ile Pro Glu 1 5 10 15 Asn Arg Val Val Ser Tyr Gln Leu SerSer Arg Ser Thr Cys Leu Lys 20 25 30 Ala Gly Val Ile Phe Thr Thr Lys LysGly Gln Gln Phe Cys Gly Asp 35 40 45 Pro Lys Gln Glu Trp Val Gln Arg TyrMet Lys Asn Leu Asp Ala Lys 50 55 60 Gln Lys Lys Ala Ser Pro Arg Ala ArgAla Val Ala Val Lys Gly Pro 65 70 75 80 Val Gln Arg Tyr Pro Gly Asn GlnThr 85 90 amino acids amino acid linear protein 113 Met Pro Ser Pro CysCys Met Phe Phe Val Ser Lys Arg Ile Pro Glu 1 5 10 15 Asn Arg Val ValSer Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys 20 25 30 Ala Gly Val IlePhe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp 35 40 45 Pro Lys Gln GluTrp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys 50 55 60 Gln Lys Lys AlaSer Pro Arg Ala Arg Ala Val Ala Val Lys Gly Pro 65 70 75 80 Val Gln ArgTyr Pro Gly Asn Gln Thr Thr 85 90 91 amino acids amino acid linearprotein 114 Met Pro Ser Pro Cys Cys Met Phe Phe Val Ser Lys Arg Ile ProGlu 1 5 10 15 Asn Arg Val Val Ser Tyr Gln Leu Ser Ser Arg Ser Thr CysLeu Lys 20 25 30 Ala Gly Val Ile Phe Thr Thr Lys Lys Gly Gln Gln Phe CysGly Asp 35 40 45 Pro Lys Gln Glu Trp Val Gln Arg Tyr Met Lys Asn Leu AspAla Lys 50 55 60 Gln Lys Lys Ala Ser Pro Arg Ala Arg Ala Val Ala Val LysGly Pro 65 70 75 80 Val Gln Arg Tyr Pro Gly Asn Gln Thr Thr Cys 85 90

What is claimed is:
 1. An isolated polynucleotide comprising a nucleicacid selected from the group consisting of: (a) a nucleic acid encodinga polypeptide comprising the amino acid sequence of residue 4 to residuem in SEQ ID NO:2, wherein m is any one of residues 48-93 of SEQ ID NO:2,and wherein said polypeptide inhibits the Chemokine Receptor-3 (CCR3)signaling pathway; (b) a nucleic acid encoding a polypeptide comprisingthe amino acid sequence of (a) except for one or more conservative aminoacid substitutions, wherein said polypeptide inhibits the ChemokineReceptor-3 (CCR3) signaling pathway; (c) a nucleic acid encoding apolypeptide comprising a sequence at least 80% identical to the aminoacid sequence of (a), wherein said polypeptide inhibits the ChemokineReceptor-3 (CCR3) signaling pathway.
 2. The isolated polynucleotide ofclaim 1, which comprises the nucleic acid of (a).
 3. The isolatedpolynucleotide of claim 1, which comprises the nucleic acid of (b). 4.The isolated polynucleotide of claim 1, which comprises the nucleic acidof (c).
 5. The isolated polynucleotide of claim 2, wherein the encodedpolypeptide further comprises a Met residue at the N-terminus.
 6. Theisolated polynucleotide of claim 2, further comprising a heterologousnucleic acid.
 7. The isolated polynucleotide of claim 6, wherein saidheterologous nucleic acid encodes a heterologous polypeptide.
 8. Amethod of producing a vector that comprises inserting the isolatedpolynucleotide of claim 2 into a vector.
 9. A vector produced by themethod of claim
 8. 10. The vector of claim 9, wherein saidpolynucleotide is operably associated with a heterologous regulatorysequence.
 11. A host cell comprising the isolated polynucleotide ofclaim
 2. 12. The host cell of claim 11, wherein said polynucleotide isoperably associated with a heterologous regulatory sequence.
 13. Amethod of producing a polypeptide comprising culturing the host cell ofclaim 12 under conditions such that the encoded polypeptide isexpressed, and recovering said encoded polypeptide.
 14. A compositioncomprising the polynucleotide of claim
 2. 15. The isolatedpolynucleotide of claim 3, wherein the encoded polypeptide furthercomprises a Met residue at the N-terminus.
 16. The isolatedpolynucleotide of claim 3, further comprising a heterologous nucleicacid.
 17. The isolated polynucleotide of claim 16, wherein saidheterologous nucleic acid encodes a heterologous polypeptide.
 18. Amethod of producing a vector that comprises inserting the isolatedpolynucleotide of claim 3 into a vector.
 19. A vector produced by themethod of claim
 18. 20. The vector of claim 19, wherein saidpolynucleotide is operably associated with a heterologous regulatorysequence.
 21. A host cell comprising the isolated polynucleotide ofclaim
 3. 22. The host cell of claim 21, wherein said polynucleotide isoperably associated with a heterologous regulatory sequence.
 23. Amethod of producing a polypeptide comprising culturing the host cell ofclaim 22 under conditions such that the encoded polypeptide isexpressed, and recovering said encoded polypeptide.
 24. A compositioncomprising the polynucleotide of claim
 3. 25. The isolatedpolynucleotide of claim 4, wherein the encoded polypeptide furthercomprises a Met residue at the N-terminus.
 26. The isolatedpolynucleotide of claim 4, further comprising a heterologous nucleicacid.
 27. The isolated polynucleotide of claim 26, wherein saidheterologous nucleic acid encodes a heterologous polypeptide.
 28. Amethod of producing a vector that comprises inserting the isolatedpolynucleotide of claim 4 into a vector.
 29. A vector produced by themethod of claim
 28. 30. The vector of claim 29, wherein saidpolynucleotide is operably associated with a heterologous regulatorysequence.
 31. A host cell comprising the isolated polynucleotide ofclaim
 4. 32. The host cell of claim 31, wherein said polynucleotide isoperably associated with a heterologous regulatory sequence.
 33. Amethod ofproducing a polypeptide comprising culturing the host cell ofclaim 32 under conditions such that the encoded polypeptide isexpressed, and recovering said encoded polypeptide.
 34. A compositioncomprising the polynucleotide of claim
 4. 35. The isolatedpolynucleotide of claim 2, wherein m is residue 48 of SEQ ID NO:2. 36.The isolated polynucleotide of claim 3, wherein m is residue 48 of SEQID NO:2.
 37. The isolated polynucleotide of claim 4, wherein m isresidue 48 of SEQ ID NO:2.
 38. The isolated polynucleotide of claim 2,wherein m is residue 73 of SEQ ID NO:2.
 39. The isolated polynucleotideof claim 3, wherein m is residue 73 of SEQ ID NO:2.
 40. The isolatedpolynucleotide of claim 4, wherein m is residue 73 of SEQ ID NO:2. 41.The isolated polynucleotide of claim 2, wherein m is residue 93 of SEQID NO:2.
 42. The isolated polynucleotide of claim 3, wherein m isresidue 93 of SEQ ID NO:2.
 43. The isolated polynucleotide of claim 4,wherein m is residue 93 of SEQ ID NO:2.
 44. The isolated polynucleotideof claim 35, wherein said nucleic acid encodes encoded polypeptidefurther comprises a Met residue at the N-terminus.
 45. The isolatedpolynucleotide of claim 36, wherein the encoded polypeptide furthercomprises a Met residue at the N-terminus.
 46. The isolatedpolynucleotide of claim 37, wherein the encoded polypeptide furthercomprises a Met residue at the N-terminus.
 47. The isolatedpolynucleotide of claim 38, wherein the encoded polypeptide furthercomprises a Met residue at the N-terminus.
 48. The isolatedpolynucleotide of claim 39, wherein the encoded polypeptide furthercomprises a Met residue at the N-terminus.
 49. The isolatedpolynucleotide of claim 40, wherein the encoded polypeptide furthercomprises a Met residue at the N-terminus.
 50. The isolatedpolynucleotide of claim 41, wherein the encoded polypeptide furthercomprises a Met residue at the N-terminus.
 51. The isolatedpolynucleotide of claim 42, wherein the encoded polypeptide furthercomprises a Met residue at the N-terminus.
 52. The isolatedpolynucleotide of claim 43, wherein the encoded polypeptide furthercomprises a Met residue at the N-terminus.
 53. The isolatedpolynucleotide of claim 4, wherein the encoded polypeptide comprises asequence at least 90% identical to the amino acid sequence of (a). 54.The isolated polynucleotide of claim 53, wherein the encoded polypeptidecomprises a sequence at least 95% identical to the amino acid sequenceof (a).
 55. An isolated polynucleotide consisting of a nucleic acidencoding an amino acid sequence shown in any one of SEQ ID NOs:23-68.56. The isolated polynucleotide of claim 55, wherein said nucleic acidencodes the amino acid sequence in SEQ ID NO:23.
 57. The isolatedpolynucleotide of claim 55, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:24.
 58. The isolated polynucleotide of claim55, wherein said nucleic acid encodes the amino acid sequence in SEQ IDNO:25.
 59. The isolated polynucleotide of claim 55, wherein said nucleicacid encodes the amino acid sequence in SEQ ID NO:26.
 60. The isolatedpolynucleotide of claim 55, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:27.
 61. The isolated polynucleotide of claim55, wherein said nucleic acid encodes the amino acid sequence in SEQ IDNO:28.
 62. The isolated polynucleotide of claim 55, wherein said nucleicacid encodes the amino acid sequence in SEQ ID NO:29.
 63. The isolatedpolynucleotide of claim 55, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:30.
 64. The isolated polynucleotide of claim55, wherein said nucleic acid encodes the amino acid sequence in SEQ IDNO:31.
 65. The isolated polynucleotide of claim 55, wherein said nucleicacid encodes the amino acid sequence in SEQ ID NO:32.
 66. The isolatedpolynucleotide of claim 55, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:33.
 67. The isolated polynucleotide of claim55, wherein said nucleic acid encodes the amino acid sequence in SEQ IDNO:34.
 68. The isolated polynucleotide of claim 55, wherein said nucleicacid encodes the amino acid sequence in SEQ ID NO:35.
 69. The isolatedpolynucleotide of claim 55, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:36.
 70. The isolated polynucleotide of claim55, wherein said nucleic acid encodes the amino acid sequence in SEQ IDNO:37.
 71. The isolated polynucleotide of claim 55, wherein said nucleicacid encodes the amino acid sequence in SEQ ID NO:38.
 72. The isolatedpolynucleotide of claim 55, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:39.
 73. The isolated polynucleotide of claim55, wherein said nucleic acid encodes the amino acid sequence in SEQ IDNO:40.
 74. The isolated polynucleotide of claim 55, wherein said nucleicacid encodes the amino acid sequence in SEQ ID NO:41.
 75. The isolatedpolynucleotide of claim 55, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:42.
 76. The isolated polynucleotide of claim55, wherein said nucleic acid encodes the amino acid sequence in SEQ IDNO:43.
 77. The isolated polynucleotide of claim 55, wherein said nucleicacid encodes the amino acid sequence in SEQ ID NO:44.
 78. The isolatedpolynucleotide of claim 55, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:45.
 79. The isolated polynucleotide of claim55, wherein said nucleic acid encodes the amino acid sequence in SEQ IDNO:46.
 80. The isolated polynucleotide of claim 55, wherein said nucleicacid encodes the amino acid sequence in SEQ ID NO:47.
 81. The isolatedpolynucleotide of claim 55, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:48.
 82. The isolated polynucleotide of claim55, wherein said nucleic acid encodes the amino acid sequence in SEQ IDNO:49.
 83. The isolated polynucleotide of claim 55, wherein said nucleicacid encodes the amino acid sequence in SEQ ID NO:50.
 84. The isolatedpolynucleotide of claim 55, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:51.
 85. The isolated polynucleotide of claim55, wherein said nucleic acid encodes the amino acid sequence in SEQ IDNO:52.
 86. The isolated polynucleotide of claim 55, wherein said nucleicacid encodes the amino acid sequence in SEQ ID NO:53.
 87. The isolatedpolynucleotide of claim 55, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:54.
 88. The isolated polynucleotide of claim55, wherein said nucleic acid encodes the amino acid sequence in SEQ IDNO:55.
 89. The isolated polynucleotide of claim 55, wherein said nucleicacid encodes the amino acid sequence in SEQ ID NO:56.
 90. The isolatedpolynucleotide of claim 55, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:57.
 91. The isolated polynucleotide of claim55, wherein said nucleic acid encodes the amino acid sequence in SEQ IDNO:58.
 92. The isolated polynucleotide of claim 55, wherein said nucleicacid encodes the amino acid sequence in SEQ ID NO:59.
 93. The isolatedpolynucleotide of claim 55, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:60.
 94. The isolated polynucleotide of claim55, wherein said nucleic acid encodes the amino acid sequence in SEQ IDNO:61.
 95. The isolated polynucleotide of claim 55, wherein said nucleicacid encodes the amino acid sequence in SEQ ID NO:62.
 96. The isolatedpolynucleotide of claim 55, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:63.
 97. The isolated polynucleotide of claim55, wherein said nucleic acid encodes the amino acid sequence in SEQ IDNO:64.
 98. The isolated polynucleotide of claim 55, wherein said nucleicacid encodes the amino acid sequence in SEQ ID NO:65.
 99. The isolatedpolynucleotide of claim 55, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:66.
 100. The isolated polynucleotide of claim55, wherein said nucleic acid encodes the amino acid sequence in SEQ IDNO:67.
 101. The isolated polynucleotide of claim 55, wherein saidnucleic acid encodes the amino acid sequence in SEQ ID NO:68.
 102. Theisolated polynucleotide of claim 55, which is fused to a heterologousnucleic acid.
 103. The isolated polynucleotide of claim 102, whereinsaid heterologous nucleic acid encodes a heterologous polypeptide. 104.A method of producing a vector that comprises inserting the isolatedpolynucleotide of claim 55 into a vector.
 105. A vector produced by themethod of claim
 104. 106. The vector of claim 105, wherein saidpolynucleotide is operably associated with a heterologous regulatorysequence.
 107. A host cell comprising the isolated polynucleotide ofclaim
 55. 108. The host cell of claim 107, wherein said polynucleotideis operably associated with a heterologous regulatory sequence.
 109. Amethod ofproducing a polypeptide comprising culturing the host cell ofclaim 108 under conditions such that the encoded polypeptide isexpressed, and recovering said encoded polypeptide.
 110. A compositioncomprising the polynucleotide of claim
 55. 111. An isolatedpolynucleotide consisting of a nucleic acid encoding an amino acidsequence shown in any one of SEQ ID NOs:69-114.
 112. The isolatedpolynucleotide of claim 111, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:69.
 113. The isolated polynucleotide of claim111, wherein said nucleic acid encodes the amino acid sequence in SEQ IDNO:70.
 114. The isolated polynucleotide of claim 111, wherein saidnucleic acid encodes the amino acid sequence in SEQ ID NO:71.
 115. Theisolated polynucleotide of claim 111, wherein said nucleic acid encodesthe amino acid sequence in SEQ ID NO:72.
 116. The isolatedpolynucleotide of claim 111, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:73.
 117. The isolated polynucleotide of claim111, wherein said nucleic acid encodes the amino acid sequence in SEQ IDNO:74.
 118. The isolated polynucleotide of claim 111, wherein saidnucleic acid encodes the amino acid sequence in SEQ ID NO:75.
 119. Theisolated polynucleotide of claim 111, wherein said nucleic acid encodesthe amino acid sequence in SEQ ID NO:76.
 120. The isolatedpolynucleotide of claim 111, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:77.
 121. The isolated polynucleotide of claim111, wherein said nucleic acid encodes the amino acid sequence in SEQ IDNO:78.
 122. The isolated polynucleotide of claim 111, wherein saidnucleic acid encodes the amino acid sequence in SEQ ID NO:79.
 123. Theisolated polynucleotide of claim 111, wherein said nucleic acid encodesthe amino acid sequence in SEQ ID NO:80.
 124. The isolatedpolynucleotide of claim 111, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:81.
 125. The isolated polynucleotide of claim111, wherein said nucleic acid encodes the amino acid sequence in SEQ IDNO:82.
 126. The isolated polynucleotide of claim 111, wherein saidnucleic acid encodes the amino acid sequence in SEQ ID NO:83.
 127. Theisolated polynucleotide of claim 111, wherein said nucleic acid encodesthe amino acid sequence in SEQ ID NO:84.
 128. The isolatedpolynucleotide of claim 111, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:85.
 129. The isolated polynucleotide of claim111, wherein said nucleic acid encodes the amino acid sequence in SEQ IDNO:86.
 130. The isolated polynucleotide of claim 111, wherein saidnucleic acid encodes the amino acid sequence in SEQ ID NO:87.
 131. Theisolated polynucleotide of claim 111, wherein said nucleic acid encodesthe amino acid sequence in SEQ ID NO:88.
 132. The isolatedpolynucleotide of claim 111, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:89.
 133. The isolated polynucleotide of claim111, wherein said nucleic acid encodes the amino acid sequence in SEQ IDNO:90.
 134. The isolated polynucleotide of claim 111, wherein saidnucleic acid encodes the amino acid sequence in SEQ ID NO:91.
 135. Theisolated polynucleotide of claim 111, wherein said nucleic acid encodesthe amino acid sequence in SEQ ID NO:92.
 136. The isolatedpolynucleotide of claim 111, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:93.
 137. The isolated polynucleotide of claim111, wherein said nucleic acid encodes the amino acid sequence in SEQ IDNO:94.
 138. The isolated polynucleotide of claim 111, wherein saidnucleic acid encodes the amino acid sequence in SEQ ID NO:95.
 139. Theisolated polynucleotide of claim 111, wherein said nucleic acid encodesthe amino acid sequence in SEQ ID NO:96.
 140. The isolatedpolynucleotide of claim 111, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:97.
 141. The isolated polynucleotide of claim111, wherein said nucleic acid encodes the amino acid sequence in SEQ IDNO:98.
 142. The isolated polynucleotide of claim 111, wherein saidnucleic acid encodes the amino acid sequence in SEQ ID NO:99.
 143. Theisolated polynucleotide of claim 111, wherein said nucleic acid encodesthe amino acid sequence in SEQ ID NO:100.
 144. The isolatedpolynucleotide of claim 111, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:101.
 145. The isolated polynucleotide ofclaim 111, wherein said nucleic acid encodes the amino acid sequence inSEQ ID NO:102.
 146. The isolated polynucleotide of claim 111, whereinsaid nucleic acid encodes the amino acid sequence in SEQ ID NO:103. 147.The isolated polynucleotide of claim 111, wherein said nucleic acidencodes the amino acid sequence in SEQ ID NO:104.
 148. The isolatedpolynucleotide of claim 111, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:105.
 149. The isolated polynucleotide ofclaim 111, wherein said nucleic acid encodes the amino acid sequence inSEQ ID NO:106.
 150. The isolated polynucleotide of claim 111, whereinsaid nucleic acid encodes the amino acid sequence in SEQ ID NO:107. 151.The isolated polynucleotide of claim 111, wherein said nucleic acidencodes the amino acid sequence in SEQ ID NO:108.
 152. The isolatedpolynucleotide of claim 111, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:109.
 153. The isolated polynucleotide ofclaim 111, wherein said nucleic acid encodes the amino acid sequence inSEQ ID NO:110.
 154. The isolated polynucleotide of claim 111, whereinsaid nucleic acid encodes the amino acid sequence in SEQ ID NO:111. 155.The isolated polynucleotide of claim 111, wherein said nucleic acidencodes the amino acid sequence in SEQ ID NO:112.
 156. The isolatedpolynucleotide of claim 111, wherein said nucleic acid encodes the aminoacid sequence in SEQ ID NO:113.
 157. The isolated polynucleotide ofclaim 111, wherein said nucleic acid encodes the amino acid sequence inSEQ ID NO:114.
 158. The isolated polynucleotide of claim 111, which isfused to a heterologous nucleic acid.
 159. The isolated polynucleotideof claim 158, wherein said heterologous nucleic acid encodes aheterologous polypeptide.
 160. A method of producing a vector thatcomprises inserting the isolated polynucleotide of claim 111 into avector.
 161. A vector produced by the method of claim
 160. 162. Thevector of claim 161, wherein said polynucleotide is operably associatedwith a heterologous regulatory sequence.
 163. A host cell comprising theisolated polynucleotide of claim
 111. 164. The host cell of claim 163,wherein said polynucleotide is operably associated with a heterologousregulatory sequence.
 165. A method ofproducing a polypeptide comprisingculturing the host cell of claim 164 under conditions such that theencoded polypeptide is expressed, and recovering said encodedpolypeptide.
 166. A composition comprising the polynucleotide of claim111.
 167. The isolated polynucleotide of claim 2, wherein said nucleicacid is a fragment of SEQ ID NO:1.
 168. The isolated polynucleotide ofclaim 3, wherein said nucleic acid is at least 70% identical to afragment of SEQ ID NO:1.
 169. The isolated polynucleotide of claim 168,wherein said nucleic acid is at least 90% identical to a fragment of SEQID NO:1.
 170. The isolated polynucleotide of claim 169, wherein saidnucleic acid is at least 95% identical to a fragment of SEQ ID NO:1.171. The isolated polynucleotide of claim 170, wherein said nucleic acidis at least 99% identical to a fragment of SEQ ID NO:1.
 172. Theisolated polynucleotide of claim 4, wherein said nucleic acid is atleast 70% identical to a fragment of SEQ ID NO:
 1. 173. The isolatedpolynucleotide of claim 172, wherein said nucleic acid is at least 90%identical to a fragment of SEQ ID NO:1.
 174. The isolated polynucleotideof claim 173, wherein said nucleic acid is at least 95% identical to afragment of SEQ ID NO:1.
 175. The isolated polynucleotide of claim 174,wherein said nucleic acid is at least 99% identical to a fragment of SEQID NO:1.
 176. The isolated polynucleotide of claim 55, wherein saidnucleic acid is a fragment of SEQ ID NO:1.
 177. The isolatedpolynucleotide of claim 111, wherein said nucleic acid consists of afragment of SEQ ID NO:1 and a Met codon at the 5′ end.
 178. Thecomplement of the polynucleotide of claim
 2. 179. The complement of thepolynucleotide of claim
 3. 180. The complement of the polynucleotide ofclaim
 4. 181. The complement of the polynucleotide of claim
 55. 182. Thecomplement of the polynucleotide of claim 111.